bluuebunny/crossref_metadata_2025 huggingface dataset:
An Eph receptor regulates integrin activity through R-Ras
Authors: Zou June X. , Wang Bingcheng , Kalo Matthew S. , Zisch Andreas H. , Pasquale Elena B. , Ruoslahti Erkki
Published: July 2002

The ability of integrins to mediate cell attachment to extracellular matrices and to blood proteins is regulated from inside the cell. Increased ligand-binding activity of integrins is critical for platelet aggregation upon blood clotting and for leukocyte extravasation to inflamed tissues. Decreased adhesion is thought to promote tumor cell invasion. R-Ras, a small intracellular GTPase, regulates the binding of integrins to their ligands outside the cell. Here we show that the Eph receptor tyrosine kinase, EphB2, can control integrin activity through R-Ras. Cells in which EphB2 is activated become poorly adherent to substrates coated with integrin ligands, and a tyrosine residue in the R-Ras effector domain is phosphorylated. The R-Ras phosphorylation and loss of cell adhesion are causally related, because forced expression of an R-Ras variant resistant to phosphorylation at the critical site made cells unresponsive to the anti-adhesive effect of EphB2. This is an unusual regulatory pathway among the small GTPases. Reduced adhesiveness induced through the Eph/R-Ras pathway may explain the repulsive effect of the Eph receptors in axonal pathfinding and may facilitate tumor cell invasion and angiogenesis.
The human natural killer cell immune synapse
Authors: Davis Daniel M. , Chiu Isaac , Fassett Marlys , Cohen George B. , Mandelboim Ofer , Strominger Jack L.
Published: July 2002

Inhibitory killer Ig-like receptors (KIR) at the surface of natural killer (NK) cells induced clustering of HLA-C at the contacting surface of target cells. In this manner, inhibitory immune synapses were formed as human NK cells surveyed target cells. At target/NK cell synapses, HLA-C/KIR distributed into rings around central patches of intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, the opposite orientation to mature murine T cell-activating synapses. This organization of protein was stable for at least 20 min. Cells could support multiple synapses simultaneously, and clusters of HLA-C moved as NK cells crawled over target cells. Clustering required a divalent metal cation, explaining how metal chelators inhibit KIR function. Surprisingly, however, formation of inhibitory synapses was unaffected by ATP depletion and the cytoskeletal inhibitors, colchicine and cytochalsins B and D. Clearly, supramolecular organization within plasma membranes is critical for NK cell immunosurveillance.
TOXCAT: A measure of transmembrane helix association in a biological membrane
Authors: Russ William P. , Engelman Donald M.
Published: July 2002

The noncovalent association of transmembrane α-helices is a fundamental event in the folding of helical membrane proteins. In this work, a system (TOXCAT) is developed for the study of transmembrane helix–helix oligomerization in a natural membrane environment. This assay uses a chimeric construct composed of the N-terminal DNA binding domain of ToxR (a dimerization-dependent transcriptional activator) fused to a transmembrane domain (tm) of interest and a monomeric periplasmic anchor (the maltose binding protein). Association of the tms results in the ToxR-mediated activation of a reporter gene encoding chloramphenicol acetyltransferase (CAT). The level of CAT expression indicates the strength of tm association. The assay distinguishes between a known dimerizing tm and a mutant in which dimerization is disrupted. In addition, modulation of the chimera concentration shows that the dimerization exhibits concentration dependence in membranes. TOXCAT also is used to select oligomeric tms from a library of randomized sequences, demonstrating the potential of this system to reveal novel oligomerization motifs. The TOXCAT system has been used to investigate glycophorin A tm-mediated dimerization. Although the overall sensitivity of glycophorin A tm dimerization to mutagenesis is found to be similar in membranes and in detergent micelles, several significant differences exist. Mutations to polar residues, which are generally disruptive in SDS, exhibit sequence specificity in membranes, demonstrating both the limitations of detergent micelles and the wider range of application of the TOXCAT system.
The C-terminal domain of the regulatory protein NOVH is sufficient to promote interaction with fibulin 1C: A clue for a role of NOVH in cell-adhesion signaling
Authors: Perbal Bernard , Martinerie Cécile , Sainson Richard , Werner Michel , He Bin , Roizman Bernard
Published: July 2002

The NOVH protein belongs to the emerging CCN [Connective tissue growth factor (CTGF), Cyr61/Cef10, nephroblastoma overexpressed gene] family of growth regulators sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Two members of the family (CYR61 and CTGF) are positive regulators of cell proliferation, whereas NOVH and two other members (ELM1 and RCOP-1) exhibit features of negative regulators of growth. The multimodular structure of these proteins suggests that their biological role(s) may depend on interactions with several factors as well as proteins constitutive of the extracellular matrix. To gain insight into the functionality of these domains, we have used a two-hybrid system to identify proteins interacting with NOVH. We report here that the C-terminal domain confers on the full-length NOVH protein the capacity to bind fibulin 1C, a protein of the extracellular matrix that interacts with several other regulators of cell adhesion. Furthermore, we show that a natural N-truncated isoform of NOVH produced by cells expressing the full-length NOVH protein also binds fibulin 1C with a high affinity, and we hypothesize that the production of truncated isoforms of NOVH (and probably of other CCN proteins) may be a critical aspect in the modulation of their biological activity. These results set the stage for a study of NOVH–fibulin 1C interactions and their potential significance in cell-adhesion signaling in normal and pathological conditions.
Modulation of CREB binding protein function by the promyelocytic (PML) oncoprotein suggests a role for nuclear bodies in hormone signaling
Authors: Doucas Vassilis , Tini Marc , Egan David A. , Evans Ronald M.
Published: July 2002

Disaggregation of the spherical nuclear bodies termed promyelocytic (PML) oncogenic domains (PODs) is a characteristic of acute promyelocytic leukemia. Here, we demonstrate that the cAMP enhancer binding protein (CREB)-binding protein (CBP) associates with PML
in vitro
and is recruited to the PODs
in vivo
. Through its association with CBP, wild-type PML dramatically stimulates nuclear receptor transcriptional activity. These results demonstrate that a fraction of CBP is compartmentalized to the POD through its association with PML and thus suggest that PML and other POD-associated proteins may play an unexpectedly broad role in aspects of transcriptional regulation and human disease.
Alanine is helix-stabilizing in both template-nucleated and standard peptide helices
Authors: Rohl Carol A. , Fiori Wayne , Baldwin Robert L.
Published: July 2002

Alanine-based peptides of defined sequence and length show measurable helix contents, allowing them to be used as a model system both for analyzing the mechanism of helix formation and for investigating the contributions of side-chain interactions to protein stability. Extensive characterization of many peptide sequences with varying amino acid contents indicates that the favorable helicity of alanine-based peptides can be attributed to the large helix-stabilizing propensity of alanine. Based on their analysis of alanine-rich sequences N-terminally linked to a synthetic helix-inducing template, Kemp and coworkers [Kemp, D. S., Boyd, J. G. & Muendel, C. C. (1991)
Nature (London)
352, 451–454; Kemp, D. S., Oslick, S. L. & Allen, T. J. (1996)
J. Am. Chem. Soc.
118, 4249–4255] argue that alanine is helix-indifferent, however, and that the favorable helix contents of alanine-based peptides must have some other explanation. Here, we show that the helix contents of template-nucleated sequences are influenced strongly by properties of the template–helix junction. A model in which the helix propensities of residues at the template–peptide junction are treated separately brings the results from alanine-based peptides and template-nucleated helices into agreement. The resulting model provides a physically plausible resolution of the discrepancies between the two systems and allows the helix contents of both template-nucleated and standard peptide helices to be predicted by using a single set of helix propensities. Helix formation in both standard peptides and template–peptide conjugates can be attributed to the large intrinsic helix-forming tendency of alanine.
Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-γ ligand troglitazone in patients with liposarcoma
Authors: Demetri George D. , Fletcher Christopher D. M. , Mueller Elisabetta , Sarraf Pasha , Naujoks Ryan , Campbell Natalee , Spiegelman Bruce M. , Singer Samuel
Published: July 2002

Agonist ligands for the nuclear receptor peroxisome proliferator-activated receptor-γ have been shown to induce terminal differentiation of normal preadipocytes and human liposarcoma cells
in vitro
. Because the differentiation status of liposarcoma is predictive of clinical outcomes, modulation of the differentiation status of a tumor may favorably impact clinical behavior. We have conducted a clinical trial for treatment of patients with advanced liposarcoma by using the peroxisome proliferator-activated receptor-γ ligand troglitazone, in which extensive correlative laboratory studies of tumor differentiation were performed. We report here the results of three patients with intermediate to high-grade liposarcomas in whom troglitazone administration induced histologic and biochemical differentiation
in vivo
. Biopsies of tumors from each of these patients while on troglitazone demonstrated histologic evidence of extensive lipid accumulation by tumor cells and substantial increases in NMR-detectable tumor triglycerides compared with pretreatment biopsies. In addition, expression of several mRNA transcripts characteristic of differentiation in the adipocyte lineage was induced. There was also a marked reduction in immunohistochemical expression of Ki-67, a marker of cell proliferation. Together, these data indicate that terminal adipocytic differentiation was induced in these malignant tumors by troglitazone. These results indicate that lineage-appropriate differentiation can be induced pharmacologically in a human solid tumor.
Estrogen receptor (ER) modulators each induce distinct conformational changes in ER α and ER β
Authors: Paige Lisa A. , Christensen Dale J. , Grøn Hanne , Norris John D. , Gottlin Elizabeth B. , Padilla Karen M. , Chang Ching-yi , Ballas Lawrence M. , Hamilton Paul T. , McDonnell Donald P. , Fowlkes Dana M.
Published: July 2002

Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs α and β and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER α and ER β ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.
A mutation in the heterotrimeric stimulatory guanine nucleotide binding protein α-subunit with impaired receptor-mediated activation because of elevated GTPase activity
Authors: Warner Dennis R. , Weinstein Lee S.
Published: July 2002

It has been reported that substitution of Arg
258
, a residue within the GTPase domain of the heterotrimeric guanine nucleotide binding protein (G protein) α-subunit (α
s
), to alanine (α
s
-R258A) results in decreased activation by receptor or aluminum fluoride (AlF
4
−
) and increased basal GDP release. Arg
258
interacts with Gln
170
in the helical domain, and, presumably, loss of this interaction between the GTPase and helical domain leads to more rapid GDP release, resulting in decreased activation by AlF
4
−
and increased thermolability. In this study, we mutate Gln
170
to alanine (α
s
-Q170A) and demonstrate that this mutant, like α
s
-R258A, has decreased activation by AlF
4
−
, increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release. However, unlike α
s
-R258A, α
s
-Q170A does not have impaired receptor-mediated activation. Therefore, this interdomain interaction is critical to maintain normal guanine nucleotide binding (and hence normal activation by AlF
4
−
) but is not important for receptor-mediated activation. In single turnover GTPase assays, the catalytic rate for GTP hydrolysis of α
s
-R258A was 14-fold higher than normal whereas that of α
s
-Q170A was unaffected. Examination of the α
s
crystal structure suggests that Arg
258
, through interactions with Glu
50
, might constrain the position of Arg
201
, a residue critical for catalyzing the GTPase reaction. This is an example of a mutation in a heterotrimeric G protein that results in an increased intrinsic GTPase activity and provides another mechanism by which G protein mutations can impair signal transduction.
Murine natural killer cells contribute to the granulomatous reaction caused by mycobacterial cell walls
Authors: Apostolou I. , Takahama Y. , Belmant C. , Kawano T. , Huerre M. , Marchal G. , Cui J. , Taniguchi M. , Nakauchi H. , Fournié J.-J. , Kourilsky P. , Gachelin G.
Published: July 2002

Mice injected with deproteinized cell walls prepared from the strain H37rv ofMycobacterium tuberculosisdevelop a granuloma-like lesion in which NKT cells are predominant. NKT cells play a primary role in the granulomatous response, because the latter does not occur in Jα281−/−mice, which miss NKT cells. The glycolipidic fraction of the cell walls is responsible for the recruitment of NKT cells; the recruiting activity is associated with fractions containing phosphatidylinositolmannosides. These results define a powerful experimental set up for studying thein vivoinduction of NKT cell responses to microbial components.
Molecular heterochrony in the early development of
Drosophila
Authors: Kim Junhyong , Kerr Jonita Q. , Min Gi-Sik
Published: July 2002

Heterochrony, the relative change of developmental timing, is one of the major modes of macroevolutionary change; it identifies temporally disassociated units of developmental evolution. Here, we report the results of a fine-scale temporal study for the expression of the developmental gene
hairy
and morphological development in three species of
Drosophila
,
D. melanogaster
,
D. simulans
, and
D. pseudoobscura
. The results suggest that between and among closely related species, temporal displacement of ontogenetic trajectory is detected even at the earliest stage of development. Overall,
D. simulans
shows the earliest expression, followed by
D. melanogaster,
and then by
D. pseudoobscura
. Setting
D. melanogaster
as the standard, we find the approximate time to full expression is accelerated by 13 min, 48 s in
D. simulans
and retarded by 24 min in
D. pseudoobscura
. Morphologically, again with
D. melanogaster
setting the standard, initiation of cellularization is faster in
D. simulans
by 15 min, 42 s; and initiation of morphogenesis is faster in
D. simulans
by 18 min, 7 s. These results seem to be consistent with the finding that the approximate time to full expression of
hairy
is accelerated by 13 min, 48 s in
D. simulans
. On the other hand, the same morphological events are delayed by 5 min, 32 s, and by 11 min, 32 s, respectively, in
D. pseudoobscura.
These delays are small, compared with the 24-min delay in full expression. The timing changes, in total, seem consistent with continuous phyletic evolution of temporal trajectories. Finally, we speculate that epigenetic interactions of
hairy
expression timing and cell-cycle timing may have led to morphological differences in the terminal system of the larvae.
Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification
Authors: Chen Jian-Jun , Rowley Janet D. , Wang San Ming
Published: July 2002

We have developed a technique called the generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI), to convert SAGE tags of 10 bases into their corresponding 3′ cDNA fragments covering hundred bases. A primer containing the 10-base SAGE tag is used as the sense primer, and a single base anchored oligo(dT) primer is used as an antisense primer in PCR, together with
Pfu
DNA polymerase. By using this approach, a cDNA fragment extending from the SAGE tag toward the 3′ end of the corresponding sequence can be generated. Application of the GLGI technique can solve two critical issues in applying the SAGE technique: one is that a longer fragment corresponding to a SAGE tag, which has no match in databases, can be generated for further studies; the other is that the specific fragment corresponding to a SAGE tag can be identified from multiple sequences that match the same SAGE tag. The development of the GLGI method provides several potential applications. First, it provides a strategy for even wider application of the SAGE technique for quantitative analysis of global gene expression. Second, a combined application of SAGE/GLGI can be used to complete the catalogue of the expressed genes in human and in other eukaryotic species. Third, it can be used to identify the 3′ cDNA sequence from any exon within a gene. It can also be used to confirm the reality of exons predicted by bioinformatic tools in genomic sequences. Fourth, a combined application of SAGE/GLGI can be applied to define the 3′ boundary of expressed genes in the genomic sequences in human and in other eukaryotic genomes.
Internal packing of helical membrane proteins
Authors: Eilers Markus , Shekar Srinivasan C. , Shieh Ted , Smith Steven O. , Fleming Patrick J.
Published: July 2002

Helix packing is important in the folding, stability, and association of membrane proteins. Packing analysis of the helical portions of 7 integral membrane proteins and 37 soluble proteins show that the helices in membrane proteins have higher packing values (0.431) than in soluble proteins (0.405). The highest packing values in integral membrane proteins originate from small hydrophobic (G and A) and small hydroxyl-containing (S and T) amino acids, whereas in soluble proteins large hydrophobic and aromatic residues have the highest packing values. The highest packing values for membrane proteins are found in the transmembrane helix–helix interfaces. Glycine and alanine have the highest occurrence among the buried amino acids in membrane proteins, whereas leucine and alanine are the most common buried residue in soluble proteins. These observations are consistent with a shorter axial separation between helices in membrane proteins. The tight helix packing revealed in this analysis contributes to membrane protein stability and likely compensates for the lack of the hydrophobic effect as a driving force for helix–helix association in membranes.
A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca
2+
pump (ACA2) located in the endoplasmic reticulum of
Arabidopsis
Authors: Hwang Ildoo , Sze Heven , Harper Jeffrey F.
Published: July 2002

The magnitude and duration of a cytosolic Ca
2+
release can potentially be altered by changing the rate of Ca
2+
efflux. In plant cells, Ca
2+
efflux from the cytoplasm is mediated by H
+
/Ca
2+
-antiporters and two types of Ca
2+
-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca
2+
-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca
2+
-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity (≈10%) and calmodulin stimulation (≈75%), as shown by Ca
2+
-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser
45
near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an
in vitro
substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser
45
(S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca
2+
-signaling pathways.
Effects of passage history and sampling bias on phylogenetic reconstruction of human influenza A evolution
Authors: Bush Robin M. , Smith Catherine B. , Cox Nancy J. , Fitch Walter M.
Published: July 2002

In this paper we determine the extent to which host-mediated
mutations and a known sampling bias affect evolutionary studies of
human influenza A. Previous phylogenetic reconstruction of influenza A
(H3N2) evolution using the hemagglutinin gene revealed an excess of
nonsilent substitutions assigned to the terminal branches of the tree.
We investigate two hypotheses to explain this observation. The first
hypothesis is that the excess reflects mutations that were either not
present or were at low frequency in the viral sample isolated from its
human host, and that these mutations increased in frequency during
passage of the virus in embryonated eggs. A set of 22 codons known to
undergo such “host-mediated” mutations showed a significant
excess of mutations assigned to branches attaching sequences from
egg-cultured (as opposed to cell-cultured) isolates to the tree. Our
second hypothesis is that the remaining excess results from sampling
bias. Influenza surveillance is purposefully biased toward sequencing
antigenically dissimilar strains in an effort to identify new variants
that may signal the need to update the vaccine. This bias produces an
excess of mutations assigned to terminal branches simply because an
isolate with no close relatives is by definition attached to the tree
by a relatively long branch. Simulations show that the magnitude of
excess mutations we observed in the hemagglutinin tree is consistent
with expectations based on our sampling protocol. Sampling bias does
not affect inferences about evolution drawn from phylogenetic analyses.
However, if possible, the excess caused by host-mediated mutations
should be removed from studies of the evolution of influenza viruses as
they replicate in their human hosts.
JNK and
decapentaplegic
signaling control adhesiveness and cytoskeleton dynamics during thorax closure in
Drosophila
Authors: Martín-Blanco Enrique , Pastor-Pareja José C. , García-Bellido Antonio
Published: July 2002

One of the fundamental events in metamorphosis in insects is the replacement of larval tissues by imaginal tissues. Shortly after pupariation the imaginal discs evaginate to assume their positions at the surface of the prepupal animal. This is a very precise process that is only beginning to be understood. In
Drosophila
, during embryonic dorsal closure, the epithelial cells push the amnioserosa cells, which contract and eventually invaginate in the body cavity. In contrast, we find that during pupariation the imaginal cells crawl over the passive larval tissue following a very accurate temporal and spatial pattern. Spreading is driven by filopodia and actin bridges that, protruding from the leading edge, mediate the stretching of the imaginal epithelia. Although interfering with JNK (Jun N-terminal kinase) and
dpp
(
decapentaplegic
) produces similar phenotypic effects suppressing closure, their effects at the cellular level are different. The loss of JNK activity alters the adhesion properties of larval cells and leads to the detachment of the imaginal and larval tissues. The absence of
dpp
signaling affects the actin cytoskeleton, blocks the emission of filopodia, and promotes the collapse of the leading edge of the imaginal tissues. Interestingly, these effects are very similar to those observed after interfering with JNK and
dpp
signaling during embryonic dorsal closure.
Protein binding and unfolding by the chaperone ClpA and degradation by the protease ClpAP
Authors: Hoskins Joel R. , Singh Satyendra K. , Maurizi Michael R. , Wickner Sue
Published: July 2002

ClpA, a bacterial member of the Clp/Hsp100 chaperone family, is an ATP-dependent molecular chaperone and the regulatory component of the ATP-dependent ClpAP protease. To study the mechanism of binding and unfolding of proteins by ClpA and translocation to ClpP, we used as a model substrate a fusion protein that joined the ClpA recognition signal from RepA to green fluorescent protein (GFP). ClpAP degrades the fusion protein
in vivo
and
in vitro
. The substrate binds specifically to ClpA in a reaction requiring ATP binding but not hydrolysis. Binding alone is not sufficient to destabilize the native structure of the GFP portion of the fusion protein. Upon ATP hydrolysis the GFP fusion protein is unfolded, and the unfolded intermediate can be sequestered by ClpA if a nonhydrolyzable analog is added to displace ATP. ATP is required for release. We found that although ClpA is unable to recognize native proteins lacking recognition signals, including GFP and rhodanese, it interacts with those same proteins when they are unfolded. Unfolded GFP is held in a nonnative conformation while associated with ClpA and its release requires ATP hydrolysis. Degradation of unfolded untagged proteins by ClpAP requires ATP even though the initial ATP-dependent unfolding reaction is bypassed. These results suggest that there are two ATP-requiring steps: an initial protein unfolding step followed by translocation of the unfolded protein to ClpP or in some cases release from the complex.
Somatic deletions in hereditary breast cancers implicate 13q21 as a putative novel breast cancer susceptibility locus
Authors: Kainu Tommi , Juo Suh-Hang Hank , Desper Richard , Schäffer Alejandro A. , Gillanders Elizabeth , Rozenblum Ester , Freas-Lutz Diana , Weaver Don , Stephan Dietrich , Bailey-Wilson Joan , Kallioniemi Olli-P. , Tirkkonen Mika , Syrjäkoski Kirsi , Kuukasjärvi Tuula , Koivisto Pasi , Karhu Ritva , Holli Kaija , Arason Adalgeir , Johannesdottir Gudrun , Bergthorsson Jon Thor , Johannsdottir Hrefna , Egilsson Valgardur , Barkardottir Rosa Björk , Johannsson Oskar , Haraldsson Karin , Sandberg Therese , Holmberg Eva , Grönberg Henrik , Olsson Håkan , Borg Åke , Vehmanen Paula , Eerola Hannaleena , Heikkilä Päivi , Pyrhönen Seppo , Nevanlinna Heli
Published: July 2002

A significant proportion of familial breast cancers cannot be explained by mutations in the
BRCA1
or
BRCA2
genes. We applied a strategy to identify predisposition loci for breast cancer by using mathematical models to identify early somatic genetic deletions in tumor tissues followed by targeted linkage analysis. Comparative genomic hybridization was used to study 61 breast tumors from 37 breast cancer families with no identified
BRCA1
or
BRCA2
mutations. Branching and phylogenetic tree models predicted that loss of 13q was one of the earliest genetic events in hereditary cancers. In a Swedish family with five breast cancer cases, all analyzed tumors showed distinct 13q deletions, with the minimal region of loss at 13q21-q22. Genotyping revealed segregation of a shared 13q21 germ-line haplotype in the family. Targeted linkage analysis was carried out in a set of 77 Finnish, Icelandic, and Swedish breast cancer families with no detected
BRCA1
and
BRCA2
mutations. A maximum parametric two-point logarithm of odds score of 2.76 was obtained for a marker at 13q21 (D13S1308, θ = 0.10). The multipoint logarithm of odds score under heterogeneity was 3.46. The results were further evaluated by simulation to assess the probability of obtaining significant evidence in favor of linkage by chance as well as to take into account the possible influence of the
BRCA2
locus, located at a recombination fraction of 0.25 from the new locus. The simulation substantiated the evidence of linkage at D13S1308 (
P
< 0.0017). The results warrant studies of this putative breast cancer predisposition locus in other populations.
Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells
Authors: Liu Biao , Rotenberg Susan A. , Mirkin Michael V.
Published: July 2002

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Cα), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators.
Endogenous expression of Müllerian inhibiting substance in early postnatal rat Sertoli cells requires multiple steroidogenic factor-1 and GATA-4-binding sites
Authors: Watanabe Koji , Clarke Trent R. , Lane Andrew H. , Wang Xinzhong , Donahoe Patricia K.
Published: July 2002

Müllerian inhibiting substance (MIS) is a key element required to complete mammalian male sex differentiation. The expression pattern of MIS is tightly regulated in fetal, neonatal, and prepubertal testes and adult ovaries and is well conserved among mammalian species. Although several factors have been shown to be essential to MIS expression, its regulatory mechanisms are not fully understood. We have examined MIS promoter activity in 2-day postnatal primary cultures of rat Sertoli cells that continue to express endogenous MIS mRNA. Using this system, we found that the region between human MIS−269 and −192 is necessary for full MIS promoter activity. We identified by DNase I footprint and electrophoretic mobility-shift analyses a distal steroidogenic factor-1 (SF-1)-binding site that is essential for full promoter activity. Mutational analysis of this new distal SF-1 site and the previously identified proximal SF-1 site showed that both are necessary for transcriptional activation. Moreover, the proximal promoter also contains multiple GATA-4-binding sites that are essential for functional promoter activity. Thus multiple SF-1- and GATA-4-binding sites in the MIS promoter are required for normal tissue-specific and developmental expression of MIS.
Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis
Authors: Kaminski Naftali , Allard John D. , Pittet Jean F. , Zuo Fengrong , Griffiths Mark J. D. , Morris David , Huang Xiaozhu , Sheppard Dean , Heller Renu A.
Published: July 2002

The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin β6 subunit (β6−/−), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.
Functional analysis of gapped microbial genomes: Amino acid metabolism of
Thiobacillus ferrooxidans
Authors: Selkov Evgeni , Overbeek Ross , Kogan Yakov , Chu Lien , Vonstein Veronika , Holmes David , Silver Simon , Haselkorn Robert , Fonstein Michael
Published: July 2002

A gapped genome sequence of the biomining bacterium
Thiobacillus ferrooxidans
strain ATCC23270 was assembled from sheared DNA fragments (3.2-times coverage) into 1,912 contigs. A total of 2,712 potential genes (ORFs) were identified in 2.6 Mbp (megabase pairs) of
Thiobacillus
genomic sequence. Of these genes, 2,159 could be assigned functions by using the WIT-Pro/EMP genome analysis system, most with a high degree of certainty. Nine hundred of the genes have been assigned roles in metabolic pathways, producing an overview of cellular biosynthesis, bioenergetics, and catabolism. Sequence similarities, relative gene positions on the chromosome, and metabolic reconstruction (placement of gene products in metabolic pathways) were all used to aid gene assignments and for development of a functional overview. Amino acid biosynthesis was chosen to demonstrate the analytical capabilities of this approach. Only 10 expected enzymatic activities, of the nearly 150 involved in the biosynthesis of all 20 amino acids, are currently unassigned in the
Thiobacillus
genome. This result compares favorably with 10 missing genes for amino acid biosynthesis in the complete
Escherichia
coli
genome. Gapped genome analysis can therefore give a decent picture of the central metabolism of a microorganism, equivalent to that of a complete sequence, at significantly lower cost.
Activation of gene expression by small molecule transcription factors
Authors: Mapp Anna K. , Ansari Aseem Z. , Ptashne Mark , Dervan Peter B.
Published: July 2002

Eukaryotic transcriptional activators are minimally comprised of a DNA binding domain and a separable activation domain; most activator proteins also bear a dimerization module. We have replaced these protein modules with synthetic counterparts to create artificial transcription factors. One of these, at 4.2 kDa, mediates high levels of DNA site-specific transcriptional activation
in vitro
. This molecule contains a sequence-specific DNA binding polyamide in place of the typical DNA binding region and a nonprotein linker in place of the usual dimerization peptide. Thus our activating region, a designed peptide, functions outside of the archetypal protein context, as long as it is tethered to DNA. Because synthetic polyamides can, in principle, be designed to recognize any specific sequence, these results represent a key step toward the design of small molecules that can up-regulate any specified gene.
Screening poly(dA/dT)
−
cDNAs for gene identification
Authors: Wang San Ming , Fears Scott C. , Zhang Lin , Chen Jian-Jun , Rowley Janet D.
Published: July 2002

Many genes expressed in the human genome have not been identified despite intensive efforts. We observed that the presence of long poly(dA/dT) sequences in the 3′ end of cDNA templates contributes significantly to this problem, because the hybrids formed randomly between poly(dA) and poly(dT) sequences of unrelated cDNA templates lead to loss of many templates in the normalization/subtraction reactions. The low abundant copies, which account for the majority of the expressed genes, are affected in particular by this phenomenon. We have developed a strategy called screening poly(dA/dT)
−
cDNAs for gene identification to overcome this obstacle. Applying this strategy can significantly enhance the efficiency of genome-wide gene identification and should have an impact on many functional genomic studies in the postgenome era.
Conserved plant genes with similarity to mammalian
de novo
DNA methyltransferases
Authors: Cao Xiaofeng , Springer Nathan M. , Muszynski Michael G. , Phillips Ronald L. , Kaeppler Shawn , Jacobsen Steven E.
Published: July 2002

DNA methylation plays a critical role in controlling states of gene activity in most eukaryotic organisms, and it is essential for proper growth and development. Patterns of methylation are established by
de novo
methyltransferases and maintained by maintenance methyltransferase activities. The Dnmt3 family of
de novo
DNA methyltransferases has recently been characterized in animals. Here we describe DNA methyltransferase genes from both
Arabidopsis
and maize that show a high level of sequence similarity to
Dnmt3
, suggesting that they encode plant
de novo
methyltransferases. Relative to all known eukaryotic methyltransferases, these plant proteins contain a novel arrangement of the motifs required for DNA methyltransferase catalytic activity. The N termini of these methyltransferases contain a series of ubiquitin-associated (UBA) domains. UBA domains are found in several ubiquitin pathway proteins and in DNA repair enzymes such as Rad23, and they may be involved in ubiquitin binding. The presence of UBA domains provides a possible link between DNA methylation and ubiquitin/proteasome pathways.
Identification of
Id4
as a regulator of
BRCA1
expression by using a ribozyme-library-based inverse genomics approach
Authors: Beger Carmela , Pierce Leigh N. , Krüger Martin , Marcusson Eric G. , Robbins Joan M. , Welcsh Piri , Welch Peter J. , Welte Karl , King Mary-Claire , Barber Jack R. , Wong-Staal Flossie
Published: July 2002

Expression of the breast and ovarian cancer susceptibility gene
BRCA1
is down-regulated in sporadic breast and ovarian
cancer cases. Therefore, the identification of genes involved in the
regulation of
BRCA1
expression might lead to new
insights into the pathogenesis and treatment of these tumors. In the
present study, an “inverse genomics” approach based on a
randomized ribozyme gene library was applied to identify cellular genes
regulating
BRCA1
expression. A ribozyme gene library
with randomized target recognition sequences was introduced into human
ovarian cancer-derived cells stably expressing a selectable marker
[enhanced green fluorescence protein (EGFP)] under the control of the
BRCA1
promoter. Cells in which
BRCA1
expression was upregulated by particular ribozymes were selected
through their concomitant increase in EGFP expression. The cellular
target gene of one ribozyme was identified to be the dominant negative
transcriptional regulator
Id4
. Modulation of
Id4
expression resulted in inversely regulated
expression of
BRCA1
. In addition, increase in
Id4
expression was associated with the ability of cells
to exhibit anchorage-independent growth, demonstrating the biological
relevance of this gene. Our data suggest that
Id4
is a
crucial gene regulating
BRCA1
expression and might
therefore be important for the
BRCA1
regulatory pathway
involved in the pathogenesis of sporadic breast and ovarian cancer.
Integrin activation controls metastasis in human breast cancer
Authors: Felding-Habermann Brunhilde , O'Toole Timothy E. , Smith Jeffrey W. , Fransvea Emilia , Ruggeri Zaverio M. , Ginsberg Mark H. , Hughes Paul E. , Pampori Nisar , Shattil Sanford J. , Saven Alan , Mueller Barbara M.
Published: July 2002

Metastasis is the primary cause of death in human breast cancer.
Metastasis to bone, lungs, liver, and brain involves dissemination of
breast cancer cells via the bloodstream and requires adhesion within
the vasculature. Blood cell adhesion within the vasculature depends on
integrins, a family of transmembrane adhesion receptors, and is
regulated by integrin activation. Here we show that integrin αvβ3
supports breast cancer cell attachment under blood flow conditions in
an activation-dependent manner. Integrin αvβ3 was found in two
distinct functional states in human breast cancer cells. The activated,
but not the nonactivated, state supported tumor cell arrest during
blood flow through interaction with platelets. Importantly, activated
αvβ3 was expressed by freshly isolated metastatic human breast
cancer cells and variants of the MDA-MB 435 human breast cancer cell
line, derived from mammary fat pad tumors or distant metastases in
severe combined immunodeficient mice. Expression of constitutively
activated mutant αvβ3
D723R
, but not
αvβ3
WT
, in MDA-MB 435 cells strongly promoted
metastasis in the mouse model. Thus breast cancer cells can exhibit a
platelet-interactive and metastatic phenotype that is controlled by the
activation of integrin αvβ3. Consequently, alterations within
tumors that lead to the aberrant control of integrin activation are
expected to adversely affect the course of human breast cancer.
Values‐intuitive rational action: the dynamic relationship of instrumental rationality and values insights as a form of social action
Authors: Woods Philip A.
Published: July 2002

This paper is concerned with the implications for conceptualizing social action which arise from a consideration of whether human beings are capable of knowing ultimate (universal, unconditionally valid) values. This issue is framed within the view that the validity of our understanding of social action is inextricably linked with the validity of our conception of humankind: the scope and variety of social action has potentialities and limitations that are inscribed by the nature of human beings qua human beings. The paper suggests an enlargement, through the addition of a proposed conceptual tool, of the framework that comprises Weber's typology of social action. It argues that the common human properties of the person in whom social action is embedded should not arbitrarily exclude questions of the genesis of values. An analytical argument is put forward through an examination of the extent to which a faculty for values insights is implicated in Weber's concept of charisma and ethical analysis of political leadership. The notion of values‐intuitive rational action is then outlined and discussed. The analytical argument is supported by theorizing from developmental psychology and examples of such action are given. Methodological difficulties in investigating the latter and the interrelationship of such action with institutional and social contexts are briefly discussed.
The Role of Affective Dispositions in Job Satisfaction and Work Strain: Comparing Collectivist and Individualist Societies
Authors: Chiu Randy K. , Kosinski Frederick A.
Published: July 2002

The individualism‐collectivism dimension cuts across the East and the West. It explains the differing values and cultural breeding that are necessary in acculturating people's affective and cognitive behaviours in accordance with the culture to which they belong. Many empirical studies have investigated the effects of personality traits on job satisfaction and work strain. The personality of individuals affects how they perceive the impact of job satisfaction and work stress. Furthermore, personality traits are influenced by cultural background. This study surveyed 626 nurses from collectivist and individualist societies to find out the possible difference in the role of affective dispositions in self‐reported job satisfaction and work strain between these two cultural orientations.
Global Cities and Developmental States: New York, Tokyo and Seoul
Authors: Child Hill Richard , Kim June Woo
Published: July 2002

The 'world city paradigm' assumes a convergence in economic base, spatial organisation and social structure among the world's major cities. However, Tokyo, capital of the world's second-largest national economy and the world's largest urban agglomeration, departs from the world city model on most salient dimensions. Seoul, centre of east Asia's second OECD member and the region's second-largest metropolis, exhibits the same anomaly. Tokyo and Seoul's departure from the world city hypothesis stem from late industrialisation and especially the relationship between industrial policy and finance institutionalised in a developmental state. Understanding Tokyo and Seoul necessitates a different conception of the world system from the globalist version of the world city argument. World cities differ from one another in many salient respects because they are lodged within a non-hegemonic and interdependent world political economy divided among differently organised national systems and regional alliances.
Relations between Deprivation and Immigrant Groups in Large Canadian Cities
Authors: Ley David , Smith Heather
Published: July 2002

With the co-existence of social polarisation and unprecedented immigration during recent years in major Canadian cities, this paper examines relationships between urban deprivation and the immigrant population in 1991, compared with 1971, the end of the era of the 'old' migration. Census tracts in Toronto, Montreal and Vancouver that experienced multiple deprivation are identified. Only two tracts in all three cities displayed the full set of indicators in 1991, and none in 1971. Indicators neither overlap, nor are as spatially contained, nor are as stable over time as has been true for cities in the US. Like northern Europe, there is evidence of a suburbanisation of deprivation, linked in particular to the diffusion of state-subsidised housing, especially in Toronto. In addition, and also like Europe, there are positive relationships with immigrant populations. But these relations are modest, and affect primarily recent arrivals and non-English-speaking groups. The implications of immigration are complex, because immigrants themselves are highly heterogeneous. Moreover, a longitudinal model of socio-spatial mobility rather than socio-spatial entrapment remained the dominant immigrant experience in Canadian cities.
Mobility and Affordability in US Housing
Authors: Strassmann W. Paul
Published: July 2002

In the US, affordability is changed by some housing policies that are set nationally and by others set locally. Since interventions tend to change rents and prices from market equilibrium levels, they usually lower the volume of transactions, hence mobility. Upon close examination, however, data from the 25 largest US cities do not support the (negative) association of mobility with intervention as strongly as do data comparing different countries with one another. National intervention affects cities uniformly; and local intervention, while important, is usually outweighed by other factors in the US. What matter most are the economic fortunes of particular industries that spur the growth of some cities and hold back others. The more households proliferate or migrate in, the more will a city develop new neighbourhoods and generate chains of moves or turnover of the entire housing stock. Variations in growth rates thus dominate differences in the degree of intervention as spurs or blocks to mobility.