bluuebunny/crossref_metadata_2025 huggingface dataset:
Density-dependent competition and selection on immune function in genetic lizard morphs
Authors: Svensson Erik , Sinervo Barry , Comendant Tosha
Published: July 2002

Density-dependent territorial interactions have been suggested to cause immunosuppression and thereby decrease fitness, but empirical support from natural populations is lacking. Data from a natural lizard population (Uta stansburiana) showed that breeding females surrounded by many territorial neighbors had suppressed immune function. Furthermore, variation in immunological condition had different effects on the fitness of the two heritable female throat-color morphs in this population. These interactive fitness effects caused correlational selection between female throat color and immune responsiveness. Population genetic theory predicts that this should have lead to the buildup and preservation of a genetic correlation between female morphotype and immunological condition. Accordingly, the throat color of a female was genetically correlated (rA= −1.36; SE = 0.55) with her daughter's immune responsiveness.
Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP)
Authors: Hashimoto Hitoshi , Shintani Norihito , Tanaka Kazuhiro , Mori Wakaba , Hirose Megumi , Matsuda Toshio , Sakaue Masaki , Miyazaki Jun-ichi , Niwa Hitoshi , Tashiro Fumi , Yamamoto Kyohei , Koga Kazumi , Tomimoto Shuhei , Kunugi Akiyoshi , Suetake Sayoko , Baba Akemichi
Published: July 2002

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been conserved remarkably during evolution and is widely expressed in the mammalian brain. In
Drosophila
, mutation of the PACAP homologue results in behavioral defects, including impaired olfaction-associated learning and changes in ethanol sensitivity. Here, we report the generation of mice lacking the PACAP gene (PACAP
−/−
). PACAP
−/−
mice were born in the expected Mendelian ratios but had a high early-mortality rate. The surviving adult PACAP
−/−
mice displayed remarkable behavioral changes; they exhibited hyperactive and explosive jumping behaviors in an open field, increased exploratory behavior, and less anxiety in the elevated plus maze, emergence, and novel-object tests. Analysis of PACAP
−/−
mice brains revealed that the serotonin metabolite 5-hydroxyindoleacetic acid was slightly decreased in the cortex and striatum compared with wild-type mice. The present study provides evidence that PACAP plays a previously uncharacterized role in the regulation of psychomotor behaviors.
Reprogramming leukemic cells to terminal differentiation by inhibiting specific cyclin-dependent kinases in G
1
Authors: Matushansky Igor , Radparvar Farshid , Skoultchi Arthur I.
Published: July 2002

Some tumor cells can be stimulated to differentiate and
undergo terminal cell division and loss of tumorigenicity. The
in vitro
differentiation of murine erythroleukemia (MEL)
cells is a dramatic example of tumor-cell reprogramming. We found that
reentry of MEL cells into terminal differentiation is accompanied by an
early transient decline in the activity of cyclin-dependant kinase
(CDK) 2, followed by a decline of CDK6. Later, as cells undergo
terminal arrest, CDK2 and CDK4 activities decline. By analyzing stable
MEL-cell transfectants containing vectors directing inducible
expression of specific CDK inhibitors, we show that only inhibitors
that block the combination of CDK2 and CDK6 trigger differentiation.
Inhibiting CDK2 and CDK4 does not cause differentiation. Importantly,
we also show that reprogramming through inhibition of CDKs is
restricted to G
1
phase of the cell cycle. The results imply
that abrogation of normal cell-cycle controls in tumor cells
contributes to their inability to differentiate fully and that
restoration of such controls in G
1
can lead to resumption
of differentiation and terminal cell division. The results also
indicate that CDK4 and CDK6 are functionally distinct and support our
hypothesis that the two CDKs regulate cell division at different stages
of erythroid maturation.
Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers.
Authors: Kukowska-Latallo J F , Bielinska A U , Johnson J , Spindler R , Tomalia D A , Baker J R
Published: July 2002

Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines.
Regulation of patched by sonic hedgehog in the developing neural tube.
Authors: Marigo V , Tabin C J
Published: July 2002

Ventral cell fates in the central nervous system are induced by Sonic hedgehog, a homolog of hedgehog, a secreted Drosophila protein. In the central nervous system, Sonic hedgehog has been identified as the signal inducing floor plate, motor neurons, and dopaminergic neurons. Sonic hedgehog is also involved in the induction of ventral cell type in the developing somites. ptc is a key gene in the Drosophila hedgehog signaling pathway where it is involved in transducing the hedgehog signal and is also a transcriptional target of the signal. PTC, a vertebrate homolog of this Drosophila gene, is genetically downstream of Sonic hedgehog (Shh) in the limb bud. We analyze PTC expression during chicken neural and somite development and find it expressed in all regions of these tissues known to be responsive to Sonic hedgehog signal. As in the limb bud, ectopic expression of Sonic hedgehog leads to ectopic induction of PTC in the neural tube and paraxial mesoderm. This conservation of regulation allows us to use PTC as a marker for Sonic hedgehog response. The pattern of PTC expression suggests that Sonic hedgehog may play an inductive role in more dorsal regions of the neural tube than have been previously demonstrated. Examination of the pattern of PTC expression also suggests that PTC may act in a negative feedback loop to attenuate hedgehog signaling.
SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form.
Authors: Gaudu P , Weiss B
Published: July 2002

SoxR protein is known to function both as a sensor and as a transcriptional activator for a superoxide response regulon in Escherichia coli. The activity of SoxR was tested by its ability to enable the transcription of its target gene, soxS, in vitro. The activity of the oxidized form was lost when its [2Fe-2S] clusters were reduced by dithionite under anaerobic conditions, and it was rapidly restored by autooxidation. This result is consistent with the hypothesis that induction of the regulon is effected by the univalent oxidation of the Fe-S centers of SoxR. In vivo, this oxidation may be caused by an alteration of the redox balance of electron chain intermediates that normally maintains soxR in an inactive, reduced state. Oxidized SoxR was about twice as effective as reduced SoxR in protecting the soxS operator from endonucleolytic cleavage. However, this difference could not account for a greater than 50-fold difference in their activities and therefore could not support a model in which oxidation activates SoxR by enabling it to bind to DNA. NADPH, ferredoxin, flavodoxin, or ferredoxin (flavodoxin):NADP+ reductase could not reduce SoxR directly in vitro at a measurable rate. The midpoint potential for SoxR was measured at -283 mV.
Inhibition of calmodulin-dependent phosphodiesterase induces apoptosis in human leukemic cells.
Authors: Jiang X , Li J , Paskind M , Epstein P M
Published: July 2002

Cytosolic extracts from a human lymphoblastoid B-cell line, RPMI-8392, established from a patient with acute lymphocytic leukemia, contain two major forms of cyclic nucleotide phosphodiesterase (PDE): Ca2+-calmodulin dependent PDE (PDE1) and cAMP-specific PDE (PDE4). In contrast, normal quiescent human peripheral blood lymphocytes (HPBL) are devoid of PDE1 activity [Epstein, P. M., Moraski, S., Jr., and Hachisu, R. (1987) Biochem. J. 243, 533-539]. Using reverse transcription-polymerase chain reaction (RT-PCR), we show that the mRNA encoding the 63-kDa form of PDE1 (PDE1B1) is expressed in RPMI-8392 cells, but not in normal, resting HPBL. This mRNA is, however, induced in HPBL following mitogenic stimulation by phytohemagglutinin (PHA). Also using RT-PCR, the full open reading frame for human PDE1B1 cDNA was cloned from RPMI-8392 cells and it encodes a protein of 536 amino acids with 96% identity to bovine, rat, and mouse species. RT-PCR also identifies the presence of PDE1B1 in other human lymphoblastoid and leukemic cell lines of B- (RPMI-1788, Daudi) and T-(MOLT-4, NA, Jurkat) cell origin. Inhibition of PDE1 or PDE4 activity by selective inhibitors induced RPMI-8392 cells, as well as the other cell lines, to undergo apoptosis. Culture of RPMI-8392 cells with an 18-bp phosphorothioate antisense oligodeoxynucleotide, targeted against the translation initiation region of the RPMI-8392 mRNA, led to a specific reduction in the amount of PDE1B1 mRNA after 1 day, and its disappearance after 2 days, and induced apoptosis in these cells in a sequence specific manner. This suggests that PDEs, particularly PDE1B1, because its expression is selective, may be useful targets for inducing the death of leukemic cells.
Developmental abnormalities and age-related neurodegeneration in a mouse model of Down syndrome
Authors: Holtzman David M. , Santucci Daniela , Kilbridge Joshua , Chua-Couzens Jane , Fontana David J. , Daniels Scott E. , Johnson Randolph M. , Chen Karen , Sun Yuling , Carlson Elaine , Alleva Enrico , Epstein Charles J. , Mobley William C.
Published: July 2002

To study the pathogenesis of central nervous system abnormalities
in Down syndrome (DS), we have analyzed a new genetic model of DS, the
partial trisomy 16 (Ts65Dn) mouse. Ts65Dn mice have an extra copy of
the distal aspect of mouse chromosome 16, a segment homologous to human
chromosome 21 that contains much of the genetic material responsible
for the DS phenotype. Ts65Dn mice show developmental delay during the
postnatal period as well as abnormal behaviors in both young and adult
animals that may be analogous to mental retardation. Though the Ts65Dn
brain is normal on gross examination, there is age-related degeneration
of septohippocampal cholinergic neurons and astrocytic hypertrophy,
markers of the Alzheimer disease pathology that is present in elderly
DS individuals. These findings suggest that Ts65Dn mice may be used to
study certain developmental and degenerative abnormalities in the DS
brain.
The fractionation of working memory
Authors: Baddeley Alan
Published: July 2002

In performing many complex tasks, it is necessary to hold
information in temporary storage to complete the task. The system used
for this is referred to as working memory. Evidence for the need to
postulate separable memory systems is summarized, and one particular
model of working memory is described, together with its fractionation
into three principal subsystems. The model has proved durable and
useful and, with the development of electrophysiological and positive
emission tomography scanning measures, is proving to map readily onto
recent neuroanatomical developments.
Cognitive and neurobiologic markers of early Alzheimer disease
Authors: Albert Marilyn S.
Published: July 2002

Recent studies indicate that impairments in two cognitive domains
characterize the cognitive abnormalities that appear earliest in the
course of Alzheimer disease (AD). These cognitive domains pertain to
memory and executive function ability; in particular, memory test
scores reflecting the difference between immediate and delayed recall
and tasks that assess cognitive flexibility (e.g., set-shifting).
Preliminary data indicate that tasks of this nature, along with
specific genetic information (i.e., APOE-4 status), are important in
identifying which individuals with recent cognitive changes (considered
to have “questionable” disease) will progress to the point where
they meet criteria for AD over time. When this cognitive and genetic
information is combined with neuroimaging measures targeted at the
brain regions demonstrating pathology early in AD, it may serve as
specific and accurate prognostic markers of AD.
Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody
Authors: Yuan Fan , Chen Yi , Dellian Marc , Safabakhsh Nina , Ferrara Napoleone , Jain Rakesh K.
Published: July 2002

The hyperpermeability of tumor vessels to macromolecules,
compared with normal vessels, is presumably due to vascular endothelial
growth factor/vascular permeability factor (VEGF/VPF) released by
neoplastic and/or host cells. In addition, VEGF/VPF is a potent
angiogenic factor. Removal of this growth factor may reduce the
permeability and inhibit tumor angiogenesis. To test these hypotheses,
we transplanted a human glioblastoma (U87), a human colon
adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two
locations in immunodeficient mice: the cranial window and the dorsal
skinfold chamber. The mice bearing vascularized tumors were treated
with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492
μg/ml) against VEGF/VPF or PBS (control). We found that tumor
vascular permeability to albumin in antibody-treated groups was lower
than in the matched controls and that the effect of the antibody was
time-dependent and influenced by the mode of injection. Tumor vascular
permeability did not respond to i.p. injection of the antibody until 4
days posttreatment. However, the permeability was reduced within 6
h after i.v. injection of the same amount of antibody. In addition to
the reduction in vascular permeability, the tumor vessels became
smaller in diameter and less tortuous after antibody injections and
eventually disappeared from the surface after four consecutive
treatments in U87 tumors. These results demonstrate that tumor vascular
permeability can be reduced by neutralization of endogenous
VEGF/VPF and suggest that angiogenesis and the maintenance of
integrity of tumor vessels require the presence of VEGF/VPF in the
tissue microenvironment. The latter finding reveals a new mechanism of
tumor vessel regression—i.e., blocking the interactions between
VEGF/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in
solid tumors causes dramatic reduction in vessel diameter, which may
block the passage of blood elements and thus lead to vascular
regression.
The TEL/platelet-derived growth factor β receptor (PDGFβR) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGFβR kinase-dependent signaling pathways
Authors: Carroll Martin , Tomasson Michael H. , Barker George F. , Golub Todd R. , Gilliland D. Gary
Published: July 2002

The TEL/PDGFβR fusion protein is the product of the t(5;12)
translocation in patients with chronic myelomonocytic leukemia. The
TEL/PDGFβR is an unusual fusion of a putative transcription factor,
TEL, to a receptor tyrosine kinase. The translocation fuses the amino
terminus of TEL, containing the helix-loop-helix (HLH) domain, to the
transmembrane and cytoplasmic domain of the PDGFβR. We hypothesized
that TEL/PDGFβR self-association, mediated by the HLH domain of
TEL, would lead to constitutive activation of the PDGFβR tyrosine
kinase domain and cellular transformation. Analysis of
in
vitro
-translated TEL/PDGFβR confirmed that the protein
self-associated and that self-association was abrogated by deletion of
51 aa within the TEL HLH domain.
In vivo
, TEL/PDGFβR
was detected as a 100-kDa protein that was constitutively
phosphorylated on tyrosine and transformed the murine hematopoietic
cell line Ba/F3 to interleukin 3 growth factor independence.
Transformation of Ba/F3 cells required the HLH domain of TEL and the
kinase activity of the PDGFβR portion of the fusion protein.
Immunoblotting demonstrated that TEL/PDGFβR associated with
multiple signaling molecules known to associate with the activated
PDGFβR, including phospholipase C γ1, SHP2, and
phosphoinositol-3-kinase. TEL/PDGFβR is a novel transforming
protein that self-associates and activates PDGFβR-dependent signaling
pathways. Oligomerization of TEL/PDGFβR that is dependent on the
TEL HLH domain provides further evidence that the HLH domain, highly
conserved among ETS family members, is a self-association motif.
Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis.
Authors: Gottlieb R A , Dosanjh A
Published: July 2002

We have shown elsewhere that acidification is an early event in apoptosis, preceding DNA cleavage. Cells expressing the most common mutation (delF508) of the cystic fibrosis transmembrane regulator (CFTR) exhibit a higher resting intracellular pH and are unable to secrete chloride and bicarbonate in response to cAMP. We hypothesized that defective acidification in cells expressing delF508 CFTR would interfere with the acidification that accompanies apoptosis, which in turn, would prevent endonuclease activation and cleavage of DNA. We therefore determined whether the function of the CFTR would affect the process of apoptosis in mouse mammary epithelial C127 cells stably transfected with the wild-type CFTR (C127/wt) or the delF508 mutation of the CFTR (C127/508). C127 cells possessed an acid endonuclease capable of DNA degradation at low pH. Sixteen hours after treatment with cycloheximide, C127/wt cells underwent cytoplasmic acidification. In contrast, C127/508 cells failed to demonstrate acidification. Furthermore, the C127/508 cells did not show nuclear condensation or DNA fragmentation detected by in situ nick-end labeling after treatment with cycloheximide or etoposide, in contrast to the characteristic features of apoptosis demonstrated by the C127/wt cells. Measurement of cell viability indicated a preservation of cell viability in C127/508 cells but not in C127/wt cells. That this resistance to the induction of apoptosis depended upon the loss of CFTR activity is shown by the finding that inhibition of the CFTR with diphenylamine carboxylate in C127/wt cells conferred similar protection. These findings suggest a role for the CFTR in acidification during the initiation of apoptosis in epithelial cells and imply that a failure to undergo programmed cell death could contribute to the pathogenesis of cystic fibrosis.
Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity.
Authors: Khleif S N , DeGregori J , Yee C L , Otterson G A , Kaye F J , Nevins J R , Howley P M
Published: July 2002

Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity.
An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus
Authors: Zhong Xiao-Ping , Krangel Michael S.
Published: July 2002

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (Eδ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by Eδand the TCR α enhancer, and that it prevents Eδfrom opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.
A model of excitation and adaptation in bacterial chemotaxis
Authors: Spiro Peter A. , Parkinson John S. , Othmer Hans G.
Published: July 2002

Bacterial chemotaxis is widely studied because of its
accessibility and because it incorporates processes that are important
in a number of sensory systems: signal transduction, excitation,
adaptation, and a change in behavior, all in response to stimuli.
Quantitative data on the change in behavior are available for this
system, and the major biochemical steps in the signal
transduction/processing pathway have been identified. We have
incorporated recent biochemical data into a mathematical model that can
reproduce many of the major features of the intracellular response,
including the change in the level of chemotactic proteins to step and
ramp stimuli such as those used in experimental protocols. The
interaction of the chemotactic proteins with the motor is not modeled,
but we can estimate the degree of cooperativity needed to produce the
observed gain under the assumption that the chemotactic proteins
interact directly with the motor proteins.
Ceramide-induced inhibition of T lymphocyte voltage-gated potassium channel is mediated by tyrosine kinases
Authors: Gulbins Erich , Szabo Ildiko , Baltzer Katharina , Lang Florian
Published: July 2002

The n-type K+channel (n-K+, Kv1.3) in lymphocytes has been recently implicated in the regulation of Fas-induced programmed cell death. Here, we demonstrate that ceramide, a lipid metabolite synthesized upon Fas receptor ligation, inhibits n-K+channel activity and induces a tyrosine phosphorylation of the Kv1.3 protein in Jurkat T lymphocytes. Tyrosine phosphorylation of the n-K+channel correlated with an activation of the Src-like tyrosine kinase p56lck upon cellular treatment with the ceramide analog C6-ceramide. Because genetic deficiency of p56lck or inhibition of Src-like tyrosine kinases by herbimycin A prevented ceramide-mediated n-K+channel inhibition and tyrosine phosphorylation, we propose a ceramide-initiated activation of p56lck resulting in tyrosine phosphorylation and inhibition of the n-K+channel protein.
Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination
Authors: Gu Yansong , Jin Shengfang , Gao Yijie , Weaver David T. , Alt Frederick W.
Published: July 2002

V(D)J recombination requires both lymphoid-specific and generally expressed enzymatic activities. All three known generally expressed activities involved in V(D)J recombination are also involved in DNA double-strand break repair (DSBR). Two of these are components of the DNA-dependent protein kinase (DNA-PK) and include Ku80 and DNA-PK catalytic subunit (DNA-PKcs); the third, XRCC4, is a protein of unknown function. The Ku70 protein is an additional component of DNA-PK; Ku70 forms a heterodimer with Ku80 to generate the DNA end-binding component of the enzyme. To test putative functions for Ku70, we have used gene-targeted mutation to generate a murine embryonic stem cell line which lacks Ku70 expression. We find that the Ku70
−/−
cells produce no detectable Ku70 and very little Ku80, suggesting a direct interrelationship between their levels. Correspondingly, these cells lack the nonspecific DNA end-binding activity associated with Ku. Significantly, the Ku70
−/−
embryonic stem cells have markedly increased sensitivity to γ-irradiation relative to Ku70
+/−
or wild-type embryonic stem cells. Furthermore, the Ku70
−/−
cells lack the ability to effectively rejoin signal and coding ends liberated in transiently introduced V(D)J recombination substrates by enforced RAG-1 and RAG-2 expression. We conclude that the Ku70 gene product is involved in DSBR and V(D)J recombination and confirm that the Ku70 gene can be classified as a member of the x-ray cross-complementation group 6 (XRCC6). Potential differences between the Ku70
−/−
and Ku80
−/−
V(D)J recombination defects are discussed.
BAP-135, a target for Bruton’s tyrosine kinase in response to B cell receptor engagement
Authors: Yang Weiyi , Desiderio Stephen
Published: July 2002

Bruton’s tyrosine kinase (Btk) is essential for B cell activation, but downstream targets of Btk have not been defined. We now describe a protein, BAP-135, that is associated with Btk in B cells and is a substrate for phosphorylation by Btk. BAP-135, which exhibits no detectable homology to known proteins, contains six occurrences of a hitherto undescribed amino acid repeat and two motifs, similar to the Src autophosphorylation site, that represent potential targets for tyrosine phosphorylation. The pleckstrin homology domain of Btk comprises the principal site of BAP-135 binding. Btk-dependent phosphorylation of BAP-135 is abolished by mutations that impair activation of Btk by Src-related kinases. Btk and BAP-135 exist in a complex before B cell antigen receptor (BCR) engagement; in response to BCR crosslinking, BAP-135 is transiently phosphorylated on tyrosine. Taken together, these observations suggest that BAP-135 may reside downstream of Btk in a signaling pathway originating at the BCR.
Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of
Pseudomonas aeruginosa
from the lung
Authors: Pier Gerald B. , Grout Martha , Zaidi Tanweer S.
Published: July 2002

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic
Pseudomonas aeruginosa
pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing
P. aeruginosa
from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30–100 times as many
P. aeruginosa
as cells lacking CFTR or expressing mutant ΔF508 CFTR protein. Purified CFTR inhibited ingestion of
P. aeruginosa
by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to
P. aeruginosa
and a synthetic peptide of this region inhibited
P. aeruginosa
internalization
in vivo,
leading to increased bacterial lung burdens. CFTR clears
P. aeruginosa
from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.
Regulation of DNA damage-induced apoptosis by the c-Abl tyrosine kinase
Authors: Yuan Zhi-Min , Huang Yinyin , Ishiko Takatoshi , Kharbanda Surender , Weichselbaum Ralph , Kufe Donald
Published: July 2002

Activation of the c-Abl protein tyrosine kinase by certain DNA-damaging agents contributes to down-regulation of Cdk2 and G
1
arrest by a p53-dependent mechanism. The present work investigates the potential role of c-Abl in apoptosis induced by DNA damage. Transient transfection studies with wild-type, but not kinase-inactive, c-Abl demonstrate induction of apoptosis. Cells that stably express inactive c-Abl exhibit resistance to ionizing radiation-induced loss of clonogenic survival and apoptosis. Cells null for c-
abl
are also impaired in the apoptotic response to ionizing radiation. We further show that cells deficient in p53 undergo apoptosis in response to expression of c-Abl and exhibit decreases in radiation-induced apoptosis when expressing inactive c-Abl. These findings suggest that c-Abl kinase regulates DNA damage-induced apoptosis.
A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors
Authors: Rattner Amir , Hsieh Jen-Chih , Smallwood Philip M. , Gilbert Debra J. , Copeland Neil G. , Jenkins Nancy A. , Nathans Jeremy
Published: July 2002

This paper describes the identification of a new family of mammalian genes that encode secreted proteins containing homology to the cysteine-rich ligand-binding domain found in the frizzled family of transmembrane receptors. The secreted frizzled-related proteins (sFRPs) are approximately 30 kDa in size, and each contains a putative signal sequence, a frizzled-like cysteine-rich domain, and a conserved hydrophilic carboxy-terminal domain. The sFRPs are not the products of differential splicing of the known frizzled genes. Glycosylphosphatidylinositol-anchored derivatives of sFRP-2 and sFRP-3 produced in transfected human embryonic kidney cells confer cell-surface binding by the
Drosophila
Wingless protein. These observations suggest that sFRPs may function
in vivo
to modulate Wnt signaling, or, alternatively, as novel ligands for as yet unidentified receptors.
C-terminal deletion mutant of pokeweed antiviral protein inhibits viral infection but does not depurinate host ribosomes
Authors: Tumer Nilgun E. , Hwang Duk-Ju , Bonness Maureen
Published: July 2002

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from
Phytolacca americana
, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. In addition to its ribosome-inactivating ability, PAP has potent antiviral activity against many plant and animal viruses, including HIV. We recently described the isolation and characterization of nontoxic PAP mutants, NT123-2, which has a point mutation (E176V) in the active site that abolishes enzymatic activity, and NT124-3, which has a nonsense mutation that results in deletion of the C-terminal 25 aa (W237Stop).
In vitro
translation of rabbit reticulocyte lysate ribosomes was inhibited by the C-terminal deletion mutant, but not by the active site mutant. We expressed both mutants in transgenic tobacco and showed that, unlike PAP or variant PAP, neither mutant is toxic to transgenic plants.
In vivo
depurination of rRNA was detected in transgenic tobacco expressing variant PAP, but not in transgenic plants expressing either the active site mutant or the C-terminal deletion mutant PAP. When extracts from transgenic plants containing the mutant PAPs were exogenously applied to tobacco leaves in the presence of potato virus X (PVX), the C-terminal deletion mutant had antiviral activity, while the active site mutant had no antiviral activity. Furthermore, transgenic plants expressing low levels of the C-terminal deletion mutant showed resistance to PVX infection, while transgenic plants expressing very high levels of the active site mutant PAP were not resistant to PVX. Our results demonstrate that an intact active site of PAP is necessary for antiviral activity, toxicity, and
in vivo
depurination of tobacco ribosomes. However, an intact active site is not sufficient for all these activities. An intact C terminus is also required for toxicity and depurination of tobacco ribosomes
in vivo
, but not for antiviral activity, suggesting that antiviral activity of PAP can be dissociated from its toxicity.
Direct and GTP-dependent interaction of ADP ribosylation factor 1 with coatomer subunit β
Authors: Zhao Liyun , Helms J. Bernd , Brügger Britta , Harter Cordula , Martoglio Bruno , Graf Roland , Brunner Josef , Wieland Felix T.
Published: July 2002

A site-directed photocrosslink approach was used to elucidate components that interact directly with ADP- ribosylation factor (ARF)-GTP during coat assembly. Two ARF mutants were generated that contain a photolabile amino acid at positions distant to each other within the ARF molecule. Here we show that one of the two positions specifically interacts with coatomer subunit β both on Golgi membranes and in isolated coat protein complex type I (COPI)-coated vesicles. Thus, a direct and GTP-dependent interaction of coatomer via β-coat protein complex (COP) with ARF is involved in the coating of COPI-coated vesicles. These data implicate a bivalent interaction of the complex with the donor membrane during vesicle formation.
5′ end maturation and RNA editing have to precede tRNA 3′ processing in plant mitochondria
Authors: Kunzmann Anja , Brennicke Axel , Marchfelder Anita
Published: July 2002

We report the characterization and partial purification of potato mitochondrial RNase Z, an endonuclease that generates mature tRNA 3′ ends. The enzyme consists of one (or more) protein(s) without RNA subunits. Products of the processing reaction are tRNA molecules with 3′ terminal hydroxyl groups and 3′ trailers with 5′ terminal phosphates. The main processing sites are located immediately 3′ to the discriminator and one nucleotide further downstream. This endonucleolytic processing at and close to the tRNA 3′ end in potato mitochondria suggests a higher similarity to the eukaryotic than to the prokaryotic tRNA 3′ processing pathway. Partial purification and separation of RNase Z from the 5′ processing activity RNase P allowed us to determine biochemical characteristics of the enzyme. The activity is stable over broad pH and temperature ranges, with peak activity at pH 8 and 30°C. Optimal concentrations for MgCl
2
and KCl are 5 mM and 30 mM, respectively. The potato mitochondrial RNase Z accepts only tRNA precursors with mature 5′ ends. The precursor for tRNA
Phe
requires RNA editing for efficient processing by RNase Z.
Antiviral agent blocks breathing of the common cold virus
Authors: Lewis J. Kathleen , Bothner Brian , Smith Thomas J. , Siuzdak Gary
Published: July 2002

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a “breathing” model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.
Pathogenic adaptation of
Escherichia coli
by natural variation of the FimH adhesin
Authors: Sokurenko Evgeni V. , Chesnokova Veronika , Dykhuizen Daniel E. , Ofek Itzhak , Wu Xue-Ru , Krogfelt Karen A. , Struve Carsten , Schembri Mark A. , Hasty David L.
Published: July 2002

Conventional wisdom regarding mechanisms of bacterial pathogenesis holds that pathogens arise by external acquisition of distinct virulence factors, whereas determinants shared by pathogens and commensals are considered to be functionally equivalent and have been ignored as genes that could become adapted specifically for virulence. It is shown here, however, that genetic variation in an originally commensal trait, the FimH lectin of type 1 fimbriae, can change the tropism of
Escherichia coli
, shifting it toward a urovirulent phenotype. Random point mutations in
fimH
genes that increase binding of the adhesin to mono-mannose residues, structures abundant in the oligosaccharide moieties of urothelial glycoproteins, confer increased virulence in the mouse urinary tract. These mutant FimH variants, however, are characterized by increased sensitivity to soluble inhibitors bathing the oropharyngeal mucosa, the physiological portal of
E. coli.
This functional trade-off seems to be detrimental for the intestinal ecology of the urovirulent
E. coli
. Thus, bacterial virulence can be increased by random functional mutations in a commensal trait that are adaptive for a pathologic environment, even at the cost of reduced physiological fitness in the nonpathologic habitat.
β-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo
Authors: Wikramanayake Athula H. , Huang Ling , Klein William H.
Published: July 2002

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal–vegetal axis in sea urchin embryos are largely unknown. Nuclear β-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that β-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that β-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal–vegetal axis. Our results also reveal similarities between the sea urchin animal–vegetal axis and the vertebrate dorsal–ventral axis, suggesting that these axes share a common evolutionary origin.
Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase
Authors: Delcommenne Marc , Tan Clara , Gray Virginia , Rue Laurent , Woodgett James , Dedhar Shoukat
Published: July 2002

Integrin-linked kinase (ILK) is an ankyrin-repeat containing serine–threonine protein kinase capable of interacting with the cytoplasmic domains of integrin β1, β2, and β3 subunits. Overexpression of ILK in epithelial cells disrupts cell–extracellular matrix as well as cell–cell interactions, suppresses suspension-induced apoptosis (also called Anoikis), and stimulates anchorage-independent cell cycle progression. In addition, ILK induces nuclear translocation of β-catenin, where the latter associates with a T cell factor/lymphocyte enhancer-binding factor 1 (TCF/LEF-1) to form an activated transcription factor. We now demonstrate that ILK activity is rapidly, but transiently, stimulated upon attachment of cells to fibronectin, as well as by insulin, in a phosphoinositide-3-OH kinase [Pi(3)K]-dependent manner. Furthermore, phosphatidylinositol(3,4,5)trisphosphate specifically stimulates the activity of ILK
in vitro
, and in addition, membrane targetted constitutively active Pi(3)K activates ILK
in vivo
. We also demonstrate here that ILK is an upstream effector of the Pi(3)K-dependent regulation of both protein kinase B (PKB/AKT) and glycogen synthase kinase 3 (GSK-3). Specifically, ILK can directly phosphorylate GSK-3
in vitro
and when stably, or transiently, overexpressed in cells can inhibit GSK-3 activity, whereas the overexpression of kinase-deficient ILK enhances GSK-3 activity. In addition, kinase-active ILK can phosphorylate PKB/AKT on serine-473, whereas kinase-deficient ILK severely inhibits endogenous phosphorylation of PKB/AKT on serine-473, demonstrating that ILK is involved in agonist stimulated, Pi(3)K-dependent, PKB/AKT activation. ILK is thus a receptor-proximal effector for the Pi(3)K-dependent, extracellular matrix and growth factor mediated, activation of PKB/AKT, and inhibition of GSK-3.
The genetics of caloric restriction in
Caenorhabditis elegans
Authors: Lakowski Bernard , Hekimi Siegfried
Published: July 2002

Low caloric intake (caloric restriction) can lengthen the life span of a wide range of animals and possibly even of humans. To understand better how caloric restriction lengthens life span, we used genetic methods and criteria to investigate its mechanism of action in the nematode
Caenorhabditis elegans
. Mutations in many genes (
eat
genes) result in partial starvation of the worm by disrupting the function of the pharynx, the feeding organ. We found that most
eat
mutations significantly lengthen life span (by up to 50%). In
C. elegans,
mutations in a number of other genes that can extend life span have been found. Two genetically distinct mechanisms of life span extension are known: a mechanism involving genes that regulate dauer formation (
age-1, daf-2, daf-16
, and
daf-28
) and a mechanism involving genes that affect the rate of development and behavior (
clk-1, clk-2, clk-3
, and
gro-1
). We find that the long life of
eat-2
mutants does not require the activity of DAF-16 and that
eat-2; daf-2
double mutants live even longer than extremely long-lived
daf-2
mutants. These findings demonstrate that food restriction lengthens life span by a mechanism distinct from that of dauer-formation mutants. In contrast, we find that food restriction does not further increase the life span of long-lived
clk-1
mutants, suggesting that
clk-1
and caloric restriction affect similar processes.
Role of brain allopregnanolone in the plasticity of γ-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery
Authors: Concas A. , Mostallino M. C. , Porcu P. , Follesa P. , Barbaccia M. L. , Trabucchi M. , Purdy R. H. , Grisenti P. , Biggio G.
Published: July 2002

The relation between changes in brain and plasma concentrations of neurosteroids and the function and structure of γ-aminobutyric acid type A (GABA
A
) receptors in the brain during pregnancy and after delivery was investigated in rats. In contrast with plasma, where all steroids increased in parallel, the kinetics of changes in the cerebrocortical concentrations of progesterone, allopregnanolone (AP), and allotetrahydrodeoxycorticosterone (THDOC) diverged during pregnancy. Progesterone was already maximally increased between days 10 and 15, whereas AP and allotetrahydrodeoxycorticosterone peaked around day 19. The stimulatory effect of muscimol on
36
Cl
−
uptake by cerebrocortical membrane vesicles was decreased on days 15 and 19 of pregnancy and increased 2 days after delivery. Moreover, the expression in cerebral cortex and hippocampus of the mRNA encoding for γ2L GABA
A
receptor subunit decreased during pregnancy and had returned to control values 2 days after delivery. Also α1,α2, α3, α4, β1, β2, β3, and γ2S mRNAs were measured and failed to change during pregnancy. Subchronic administration of finasteride, a 5α-reductase inhibitor, to pregnant rats reduced the concentrations of AP more in brain than in plasma as well as prevented the decreases in both the stimulatory effect of muscimol on
36
Cl
−
uptake and the decrease of γ2L mRNA observed during pregnancy. These results indicate that the plasticity of GABA
A
receptors during pregnancy and after delivery is functionally related to fluctuations in endogenous brain concentrations of AP whose rate of synthesis/metabolism appears to differ in the brain, compared with plasma, in pregnant rats.
Altered thymic positive selection and intracellular signals in Cbl-deficient mice
Authors: Naramura Mayumi , Kole Hemanta K. , Hu Ren-Ju , Gu Hua
Published: July 2002

Cbl is the product of the protooncogene c-cbland is involved in T cell antigen receptor (TCR)-mediated signaling. To understand the role of Cbl for immune system development and function, we generated a Cbl-deficient mouse strain. In Cbl-deficient mice, positive selection of the thymocytes expressing major histocompatibility complex class II-restricted transgenic TCR was significantly enhanced. Two factors may have contributed to the altered thymic selection. First, Cbl deficiency markedly up-regulated the activity of ZAP-70 and mitogen-activated protein kinases. The mitogen-activated protein kinase pathway was shown previously to be involved in thymic positive selection. Second, Cbl-deficient thymocytes expressed CD3 and CD4 molecules at higher levels, which consequently may increase the avidity of TCR/major histocompatibility complex/coreceptor interaction. Thus, Cbl plays a novel role in modulating TCR-mediated multiple signaling pathways and fine-tunes the signaling threshold for thymic selection.
A simplified system for generating recombinant adenoviruses
Authors: He Tong-Chuan , Zhou Shibin , da Costa Luis T. , Yu Jian , Kinzler Kenneth W. , Vogelstein Bert
Published: July 2002

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.