bluuebunny/crossref_metadata_2025 huggingface dataset:
The nuclear receptor
NR2E3
plays a role in human retinal photoreceptor differentiation and degeneration
Authors: Milam Ann H. , Rose Linda , Cideciyan Artur V. , Barakat Mark R. , Tang Wai-Xing , Gupta Nisha , Aleman Tomas S. , Wright Alan F. , Stone Edwin M. , Sheffield Val C. , Jacobson Samuel G.
Published: July 2002

Normal human retinal development involves orderly generation of
rods and cones by complex mechanisms. Cell-fate specification involves
progenitor cell lineage and external signals such as soluble factors
and cell–cell interactions. In most inherited human retinal
degenerations, including retinitis pigmentosa, a mutant gene causes
loss of visual function, death of mature rods, and eventually death of
all cone subtypes. Only one inherited retinal disorder, the enhanced S
cone syndrome (ESCS), shows increased visual function, involving the
minority S (blue) cones, and decreased rod and L/M (red/green) cone
function. This autosomal recessive disease is caused by mutations in
NR2E3
, a photoreceptor nuclear receptor transcription
factor, and may result from abnormal cell-fate determination, leading
to excess S cones at the expense of other photoreceptor subtypes. In 16
ESCS patients with the most common
NR2E3
mutation,
R311Q, we documented an abnormal ratio of S to L/M cone function and
progressive retinal degeneration. We studied the postmortem retina of
an ESCS patient homozygous for
NR2E3
R311Q. No rods were
identified, but cones were increased approximately 2-fold, and 92%
were S cones. Only 15% of the cones expressed L/M cone opsin, and
some coexpressed S cone opsin. The retina was disorganized, with
densely packed cones intermixed with inner retinal neurons. The retina
was also degenerate, retaining photoreceptors in only the central and
far peripheral regions. These observations suggest a key role for
NR2E3
in regulation of human photoreceptor development.
Degeneration of the
NR2E3
retina may result from
defective development, known S cone fragility, or abnormal maintenance
of mature photoreceptors.
Reduction of atherosclerosis in apolipoprotein E knockout mice by activation of the retinoid X receptor
Authors: Claudel Thierry , Leibowitz Mark D. , Fiévet Catherine , Tailleux Anne , Wagner Brandee , Repa Joyce J. , Torpier Gérard , Lobaccaro Jean-Marc , Paterniti James R. , Mangelsdorf David J. , Heyman Richard A. , Auwerx Johan
Published: July 2002

A common feature of many metabolic pathways is their control by retinoid X receptor (RXR) heterodimers. Dysregulation of such metabolic pathways can lead to the development of atherosclerosis, a disease influenced by both systemic and local factors. Here we analyzed the effects of activation of RXR and some of its heterodimers in apolipoprotein E −/− mice, a well established animal model of atherosclerosis. An RXR agonist drastically reduced the development of atherosclerosis. In addition, a ligand for the peroxisome proliferator-activated receptor (PPAR)γ and a dual agonist of both PPARα and PPARγ had moderate inhibitory effects. Both RXR and liver X receptor (LXR) agonists induced ATP-binding cassette protein 1 (ABC-1) expression and stimulated ABC-1-mediated cholesterol efflux from macrophages from wild-type, but not from LXRα and β double −/−, mice. Hence, activation of ABC-1-mediated cholesterol efflux by the RXR/LXR heterodimer might contribute to the beneficial effects of rexinoids on atherosclerosis and warrant further evaluation of RXR/LXR agonists in prevention and treatment of atherosclerosis.
Chip interacts with diverse homeodomain proteins and potentiates Bicoid activity
in vivo
Authors: Torigoi Emi , Bennani-Baïti Idriss M. , Rosen Christina , Gonzalez Katie , Morcillo Patrick , Ptashne Mark , Dorsett Dale
Published: July 2002

The
Drosophila
protein Chip potentiates activation
by several enhancers and is required for embryonic segmentation. Chip
and its mammalian homologs interact with and promote dimerization of
nuclear LIM proteins. No known
Drosophila
LIM proteins,
however, are required for segmentation, nor for expression of most
genes known to be regulated by Chip. Here we show that Chip also
interacts with diverse homeodomain proteins using residues distinct
from those that interact with LIM proteins, and that Chip potentiates
activity of one of these homeodomain proteins in
Drosophila
embryos and in yeast. These and other
observations help explain the roles of Chip in segmentation and suggest
a model to explain how Chip potentiates activation by diverse
enhancers.
A cofactor approach to copper-dependent catalytic antibodies
Authors: Nicholas Kenneth M. , Wentworth Paul , Harwig Curtis W. , Wentworth Anita D. , Shafton Asher , Janda Kim D.
Published: July 2002

A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, Lys
H93
, that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from
N
-methyl imidazole. The 38C2–5-Cu conjugate was prepared by either (
i
) initial derivatization of 38C2 with 5 followed by metallation with CuCl
2
, or (
ii
) precoordination of 5 with CuCl
2
followed by conjugation with 38C2. The resulting 38C2–5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis–Menten kinetics [
k
cat
(11) = 2.3 min
−1
and
K
m
(11) 2.2 mM] with a rate enhancement [
k
cat
(11)
k
uncat
(11)] of 2.1 × 10
5
. Comparison of the second-order rate constants of the modified 38C2 and the Cu(II)-bis-imidazolyl complex
k
(6-CuCl
2
) gives a rate enhancement of 3.5 × 10
4
in favor of the antibody complex with an effective molarity of 76.7 M, revealing a significant catalytic benefit to the binding of the bis-imidazolyl ligand into 38C2.
Tumor metastasis suppressor nm23H1 regulates Rac1 GTPase by interaction with Tiam1
Authors: Otsuki Yoshiro , Tanaka Masamitsu , Yoshii Shigeto , Kawazoe Nobuko , Nakaya Kazuyasu , Sugimura Haruhiko
Published: July 2002

The putative tumor metastasis suppressor nm23H1 was originally
identified in murine melanomas by subtraction cloning. It displays
nucleoside diphosphate kinase activity and regulates cellular events,
including growth and development. Recently nm23H1 has been reported to
also act as a GTPase-activating protein of the Ras-related GTPase Rad.
We attempted to determine whether nm23H1 also regulates Rho-family
GTPases. Although we were unable to detect a direct association between
nm23H1 and Rho-family GTPases, nm23H1 was shown to be associated with a
Rac1-specific nucleotide exchange factor, Tiam1, by interaction with
its amino-terminal region in extracts from the cells expressing
exogenous Tiam1 and from native tissue. Overexpression of nm23H1
inhibited the Tiam1-induced production of GTP-bound Rac1 and activation
of c-Jun kinase. On the other hand, forced overexpression of the wild
type, but not the kinase-inactivated mutant of nm23H1, converted the
GDP-bound forms of Rac1, Cdc42, and RhoA to their GTP-bound forms
in vitro
by its nucleoside diphosphate kinase activity,
but nm23H1 alone apparently did not produce the GTP-bound form of these
GTPases
in vivo
. These results suggest that nm23H1
negatively regulates Tiam1 and inhibits Rac1 activation
in
vivo
. Moreover, adhesion-stimulated membrane ruffles of Rat1
fibroblasts were reduced by overexpression of nm23H1. Based on these
observations, we concluded that we had identified a function of nm23H1
as a regulator of Rac1 and that it may be related to the effect of
nm23H1 as a tumor metastasis suppressor.
Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A
Authors: Bosco Daryl A. , Eisenmesser Elan Z. , Pochapsky Susan , Sundquist Wesley I. , Kern Dorothee
Published: July 2002

Packaging of cyclophilin A (CypA) into HIV-1 virions is essential for efficient replication; however, the reason for this is unknown. Incorporation is mediated through binding to the Gly-89–Pro-90 peptide bond of the N-terminal domain of HIV-1 capsid (CA
N
). Despite the fact that CypA is a peptidyl-prolyl cis/trans isomerase, catalytic activity on CA
N
has not been observed previously. We show here, using NMR exchange spectroscopy, that CypA does not only bind to CA
N
but also catalyzes efficiently the cis/trans isomerization of the Gly-89–Pro-90 peptide bond. In addition, conformational changes in CA
N
distal to the CypA binding loop are observed on CypA binding and catalysis. The results provide experimental evidence for efficient CypA catalysis on a natively folded and biologically relevant protein substrate.
ATP bound to the origin recognition complex is important for preRC formation
Authors: Klemm Richard D. , Bell Stephen P.
Published: July 2002

The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP
in vitro
. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.
Chemokine receptor 2 serves an early and essential role in resistance toMycobacterium tuberculosis
Authors: Peters Wendy , Scott Holly M. , Chambers Henry F. , Flynn JoAnne L. , Charo Israel F. , Ernst Joel D.
Published: July 2002

Although the protective cellular immune response toMycobacterium tuberculosisrequires recruitment of macrophages and T lymphocytes to the site of infection, the signals that regulate this trafficking have not been defined. We investigated the role of C-C chemokine receptor 2 (CCR2)-dependent cell recruitment in the protective response toM. tuberculosis. CCR2−/−mice died early after infection and had 100-fold more bacteria in their lungs than didCCR2+/+mice.CCR2−/−mice exhibited an early defect in macrophage recruitment to the lung and a later defect in recruitment of dendritic cells and T cells to the lung.CCR2−/−mice also had fewer macrophages and dendritic cells recruited to the mediastinal lymph node (MLN) after infection. T cell migration through the MLN was similar inCCR2−/−andCCR2+/+mice. However, T cell priming was delayed in the MLNs of theCCR2−/−mice, and fewer CD4+and CD8+T cells primed to produce IFN-γ accumulated in the lungs of theCCR2−/−mice. These data demonstrate that cellular responses mediated by activation of CCR2 are essential in the initial immune response and control of infection withM. tuberculosis.
A
RUNX2/PEBP2
αA/
CBFA1
mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia
Authors: Zhang Yu-Wen , Yasui Natsuo , Ito Kosei , Huang Gang , Fujii Makiko , Hanai Jun-ichi , Nogami Hiroshi , Ochi Takahiro , Miyazono Kohei , Ito Yoshiaki
Published: July 2002

Cleidocranial dysplasia (CCD), an autosomal-dominant human bone disease, is thought to be caused by heterozygous mutations in
runt
-related gene 2 (
RUNX2)/polyomavirus enhancer binding protein 2α
A (PEBP2α
A)/core-binding factor A1 (CBFA1)
. To understand the mechanism underlying the pathogenesis of CCD, we studied a novel mutant of
RUNX2, CCDα
A376
, originally identified in a CCD patient. The nonsense mutation, which resulted in a truncated RUNX2 protein, severely impaired RUNX2 transactivation activity. We show that signal transducers of transforming growth factor β superfamily receptors, Smads, interact with RUNX2
in vivo
and
in vitro
and enhance the transactivation ability of this factor. The truncated RUNX2 protein failed to interact with and respond to Smads and was unable to induce the osteoblast-like phenotype in C2C12 myoblasts on stimulation by bone morphogenetic protein. Therefore, the pathogenesis of CCD may be related to the impaired Smad signaling of transforming growth factor β/bone morphogenetic protein pathways that target the activity of RUNX2 during bone formation.
Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in
Arabidopsis thaliana
Authors: Awai Koichiro , Maréchal Eric , Block Maryse A. , Brun Delphine , Masuda Tatsuru , Shimada Hiroshi , Takamiya Ken-ichiro , Ohta Hiroyuki , Joyard Jacques
Published: July 2002

In
Arabidopsis
, monogalactosyldiacylglycerol (MGDG) is synthesized by a multigenic family of MGDG synthases consisting of two types of enzymes differing in their N-terminal portion: type A (atMGD1) and type B (atMGD2 and atMGD3). The present paper compares type B isoforms with the enzymes of type A that are known to sit in the inner membrane of plastid envelope. The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways. Type A
atMGD1
gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme. Its mature polypeptide is immunodetected in the envelope of chloroplasts from
Arabidopsis
leaves after cleavage of its transit peptide. atMGD1 is therefore likely devoted to the massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network. Transient expression of green fluorescent protein fusions in
Arabidopsis
leaves and
in vitro
import experiments show that type B precursors are targeted to plastids, owing to a different mechanism. Noncanonical addressing peptides, whose processing could not be assessed, are involved in the targeting of type B precursors, possibly to the outer envelope membrane where they might contribute to membrane expansion. Expression of type B enzymes was higher in nongreen tissues, i.e., in inflorescence (
atMGD2
) and roots (
atMGD3
), where they conceivably influence the eukaryotic structure prominence in MGDG. In addition, their expression of type B enzymes is enhanced under phosphate deprivation.
In vivo
mechanism-based inactivation of
S
-adenosylmethionine decarboxylases from
Escherichia coli
,
Salmonella typhimurium
, and
Saccharomyces
cerevisiae
Authors: Li Yong-Fu , Hess Sonja , Pannell Lewis K. , Tabor Celia White , Tabor Herbert
Published: July 2002

S
-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form α and β subunits. The α subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an
Escherichia coli
overexpression system, we have purified AdoMetDCs encoded by the
E. coli
,
Saccharomyces cerevisiae
, and
Salmonella typhimurium
genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally
in vivo
by a mechanism-based “suicide” inactivation. A large percentage of the α subunit of each enzyme had been modified
in vivo
to give peaks with masses
m
/
z
= 57 ± 1 and
m
/
z
= 75 ± 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the α subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 ± 1 adduct, which is probably derived from the reaction product. Comparable modification of the α subunit was also observed in
in vitro
experiments after incubation with the substrate or with the reaction product, which is consistent with the
in vitro
alkylation of
E. coli
AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991)
Biochemistry
30, 4078–4081].
Old Yellow Enzyme: Stepwise reduction of nitro-olefins and catalysis of aci-nitro tautomerization
Authors: Meah Younus , Massey Vincent
Published: July 2002

The Old Yellow Enzyme has been shown to catalyze efficiently the
NADPH-linked reduction of nitro-olefins. The reduction of the
nitro-olefin proceeds in a stepwise fashion, with formation of a
nitronate intermediate that is freely dissociable from the enzyme. The
first step involves hydride transfer from the enzyme-reduced flavin to
carbon 2 of the nitro-olefin. The protonation of the nitronate at
carbon 1 to form the final nitroalkane product also is catalyzed by the
enzyme and involves Tyr-196 as an active site acid/base. This residue
also is involved in aci-nitro tautomerization of nitroalkanes, the
first example of a nonredox reaction catalyzed by the enzyme.
Global differential gene expression in response to growth temperature alteration in group A
Streptococcus
Authors: Smoot Laura M. , Smoot James C. , Graham Morag R. , Somerville Greg A. , Sturdevant Daniel E. , Migliaccio Cristi A. Lux , Sylva Gail L. , Musser James M.
Published: July 2002

Pathogens are exposed to different temperatures during an infection cycle and must regulate gene expression accordingly. However, the extent to which virulent bacteria alter gene expression in response to temperatures encountered in the host is unknown. Group A
Streptococcus
(GAS) is a human-specific pathogen that is responsible for illnesses ranging from superficial skin infections and pharyngitis to severe invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS survives and multiplies at different temperatures during human infection. DNA microarray analysis was used to investigate the influence of temperature on global gene expression in a serotype M1 strain grown to exponential phase at 29°C and 37°C. Approximately 9% of genes were differentially expressed by at least 1.5-fold at 29°C relative to 37°C, including genes encoding transporter proteins, proteins involved in iron homeostasis, transcriptional regulators, phage-associated proteins, and proteins with no known homologue. Relatively few known virulence genes were differentially expressed at this threshold. However, transcription of 28 genes encoding proteins with predicted secretion signal sequences was altered, indicating that growth temperature substantially influences the extracellular proteome. TaqMan real-time reverse transcription–PCR assays confirmed the microarray data. We also discovered that transcription of genes encoding hemolysins, and proteins with inferred roles in iron regulation, transport, and homeostasis, was influenced by growth at 40°C. Thus, GAS profoundly alters gene expression in response to temperature. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many unforeseen lines of pathogenesis investigation.
Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in
Aplysia
Authors: Levenson Jonathan , Endo Shogo , Kategaya Lorna S. , Fernandez Raymond I. , Brabham David G. , Chin Jeannie , Byrne John H. , Eskin Arnold
Published: July 2002

An increase in transmitter release accompanying long-term
sensitization and facilitation occurs at the glutamatergic sensorimotor
synapse of
Aplysia.
We report that a long-term increase
in neuronal Glu uptake also accompanies long-term sensitization.
Synaptosomes from pleural-pedal ganglia exhibited sodium-dependent,
high-affinity Glu transport. Different treatments that induce long-term
enhancement of the siphon-withdrawal reflex, or long-term synaptic
facilitation increased Glu uptake. Moreover, 5-hydroxytryptamine, a
treatment that induces long-term facilitation, also produced a
long-term increase in Glu uptake in cultures of sensory neurons. The
mechanism for the increase in uptake is an increase in the
V
max
of transport. The long-term increase in
Glu uptake appeared to be dependent on mRNA and protein synthesis, and
transport through the Golgi, because 5,6-dichlorobenzimidazole
riboside, emetine, and brefeldin A inhibited the increase in Glu
uptake. Also, injection of emetine and 5,6-dichlorobenzimidazole into
Aplysia
prevented long-term sensitization. Synthesis of Glu
itself may be regulated during long-term sensitization because the same
treatments that produced an increase in Glu uptake also produced a
parallel increase in Gln uptake. These results suggest that coordinated
regulation of a number of different processes may be required to
establish or maintain long-term synaptic facilitation.
Peripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk) and Cdc2
Authors: Lin Chin-Yo , Madsen Matthew L. , Yarm Frederic R. , Jang Young-Joo , Liu Xiaoqi , Erikson Raymond L.
Published: July 2002

Cell division is characterized by orchestrated events of chromosome
segregation, distribution of cellular organelles, and the eventual
partitioning and separation of the two daughter cells. Mitotic kinases,
including polo-like kinases (Plk), influence multiple events in
mitosis. In yeast two-hybrid screens using mammalian Plk C-terminal
domain baits, we have identified Golgi peripheral protein GRASP65
(Golgi reassembly stacking protein of 65 kDa) as a Plk-binding protein.
GRASP65 appears to function in the postmitotic reassembly of Golgi
stacks. In this report we demonstrate binding between Plk and GRASP65
and provide
in vitro
and
in vivo
evidence
that Plk is a GRASP65 kinase. Moreover, we show that Cdc2 can also
phosphorylate GRASP65. In addition, we present data which support the
observation that the conserved C terminus of Plk is important for its
function. Deletion or frameshift mutations in the conserved C-terminal
domain of Plk greatly diminish its ability to phosphorylate GRASP65.
These and previous findings suggest that phosphorylation of Golgi
components by mitotic kinases may regulate mechanisms of Golgi
inheritance during cell division.
A hallmark of active class switch recombination: Transcripts directed by I promoters on looped-out circular DNAs
Authors: Kinoshita Kazuo , Harigai Motoko , Fagarasan Sidonia , Muramatsu Masamichi , Honjo Tasuku
Published: July 2002

To specify when and where Ig class switch recombination (CSR) takes place, a good molecular marker closely associated with active CSR is required. CSR is accompanied by deletion of circular DNA from the Ig heavy chain locus. The circular DNA contains a DNA segment between Sμ and a target S region including its I promoter, which is driven by specific cytokine stimulation before CSR. We found that the specific I promoter is still active in looped-out circular DNA and directs production of I-Cμ transcripts termed “circle transcripts.” Reverse transcription–PCR demonstrated transient induction of specific circle transcripts upon CSR in a murine lymphoma cell line, CH12F3-2A, as well as spleen B cells. Production of the circle transcripts appeared to depend on expression of activation-induced cytidine deaminase (AID), an essential factor for CSR. A comparison of kinetics between circle transcripts and circular DNA showed more rapid disappearance of circle transcripts. Thus, circle transcripts may serve as a hallmark for active CSRin vitroandin vivo.
Posttranslational modification of TEL and TEL/AML1 by SUMO-1 and cell-cycle-dependent assembly into nuclear bodies
Authors: Chakrabarti Subhra Ranjan , Sood Rashmi , Nandi Suvobroto , Nucifora Giuseppina
Published: July 2002

The E-26 transforming specific (ETS)-related gene,
TEL
, also known as
ETV6
, encodes a strong
transcription repressor that is rearranged in several recurring
chromosomal rearrangements associated with leukemia and congenital
fibrosarcoma. TEL is a nuclear phosphoprotein that is widely expressed
in all normal tissues. TEL contains a DNA-binding domain at the C
terminus and a helix–loop–helix domain (also called a pointed domain)
at the N terminus. The pointed domain is necessary for homotypic
dimerization and for interaction with the ubiquitin-conjugating enzyme
UBC9. Here we show that the interaction with UBC9 leads to modification
of TEL by conjugating it to SUMO-1. The SUMO-1-modified TEL localizes
to cell-cycle-specific nuclear speckles that we named TEL bodies. We
also show that the leukemia-associated fusion protein TEL/AML1 is
modified by SUMO-1 and found in the TEL bodies, in a pattern quite
different from what we observe and report for AML1. Therefore, SUMO-1
modification of TEL could be a critical signal necessary for normal
functioning of the protein. In addition, the modification by SUMO-1 of
TEL/AML1 could lead to abnormal localization of the fusion protein,
which could have consequences that include contribution to neoplastic
transformation.
Elevated expression of
axin2
and
hnkd
mRNA provides evidence that Wnt/β-catenin signaling is activated in human colon tumors
Authors: Yan Dong , Wiesmann Marion , Rohan Michael , Chan Vivien , Jefferson Ann B. , Guo Lida , Sakamoto Doreen , Caothien Roger H. , Fuller John H. , Reinhard Christoph , Garcia Pablo D. , Randazzo Filippo M. , Escobedo Jaime , Fantl Wendy J. , Williams Lewis T.
Published: July 2002

Genetic studies have identified mutations in key regulators of the
Wnt/β-catenin pathway in a variety of cancers, most
frequently in colon cancers. However, whether the pathway is activated
in clinical cancer samples is not easily determined, and therefore it
is useful to find markers that could be surrogates to show activation
of the Wnt/β-catenin pathway. Gene expression profiles were
analyzed in SW620, a colon cancer cell line in which β-catenin levels
are stabilized as a consequence of truncated adenomatous polyposis coli
and were compared with profiles of the same cells transfected with
antisense oligodeoxynucleotides. Treatment of cells with β-catenin
antisense oligodeoxynucleotides resulted in a decrease in the levels of
axin2
and human naked cuticle (
hnkd
)
mRNAs. Interestingly, the proteins encoded by both of these mRNAs are
known inhibitors of the β-catenin pathway. In 30 human cell lines
derived from different origins,
axin2
and
hnkd
were expressed only in human colon cancer cell
lines that are known to have activating mutations in the
Wnt/β-catenin pathway. Further, levels of both
axin2
and
hnkd
mRNA were also found to be
elevated in about 65% of laser microdissected cells from human colon
tumors compared with laser microdissected cells of normal morphology
from the same patient samples. The increased expression of
axin2
and
hnkd
correlated with
truncations in adenomatous polyposis coli in the same patient samples.
These results reveal that it is possible to detect activation of a
carcinogenic pathway in human cancer samples with specific markers.
On the role of antigen in maintaining cytotoxic T-cell memory.
Authors: Kündig T M , Bachmann M F , Oehen S , Hoffmann U W , Simard J J , Kalberer C P , Pircher H , Ohashi P S , Hengartner H , Zinkernagel R M
Published: July 2002

This study evaluated whether T-cell memory reflects increased precursor frequencies of specific long-lived T cells and/or a low-level immune response against some form of persistent antigen. Antivirally protective CD8+ T-cell memory was analyzed mostly in the original vaccinated host to assess the role of antigen in its maintenance. T-cell mediated resistance against reinfection was measured in the spleen and in peripheral solid organs with protocols that excluded protection by antibodies. In vivo protection was compared with detectable cytotoxic T-lymphocyte precursor frequencies determined in vitro. In the spleen, in vitro detectable cytotoxic T-lymphocyte precursor frequencies remained stable independently of antigen, conferring resistance against viral replication in the spleen during reinfection. In contrast, T-cell mediated resistance against reinfection of peripheral solid organs faded away in an antigen-dependent fashion within a few days or weeks. We show that only memory T cells persistently or freshly activated with antigen efficiently extravasate into peripheral organs, where cytotoxic T lymphocytes must be able to exert effector function immediately; both the capacity to extravasate and to rapidly exert effector function critically depend on restimulation by antigen. Our experiments document that the duration of T-cell memory protective against peripheral reinfection depended on the antigen dose used for immunization, was prolonged when additional antigen was provided, and was abrogated after removal of antigen. We conclude that T-cell mediated protective immunity against the usual peripheral routes of reinfection is antigen-dependent.
Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals.
Authors: Bergey D R , Howe G A , Ryan C A
Published: July 2002

The activation of plant defensive genes in leaves of tomato plants in response to herbivore damage or mechanical wounding is mediated by a mobile 18-amino acid polypeptide signal called systemin. Systemin is derived from a larger, 200-amino acid precursor called prosystemin, similar to polypeptide hormones and soluble growth factors in animals. Systemin activates a lipid-based signaling cascade, also analogous to signaling systems found in animals. In plants, linolenic acid is released from membranes and is converted to the oxylipins phytodienoic acid and jasmonic acid through the octadecanoid pathway. Plant oxylipins are structural analogs of animal prostaglandins which are derived from arachidonic acid in response to various signals, including polypeptide factors. Constitutive overexpression of the prosystemin gene in transgenic tomato plants resulted in the overproduction of prosystemin and the abnormal release of systemin, conferring a constitutive overproduction of several systemic wound-response proteins (SWRPs). The data indicate that systemin is a master signal for defense against attacking herbivores. The same defensive proteins induced by wounding are synthesized in response to oligosaccharide elicitors that are generated in leaf cells in response to pathogen attacks. Inhibitors of the octadecanoid pathway, and a mutation that interrupts this pathway, block the induction of SWRPs by wounding, systemin, and oligosaccharide elicitors, indicating that the octadecanoid pathway is essential for the activation of defense genes by all of these signals. The tomato mutant line that is functionally deficient in the octadecanoid pathway is highly susceptible to attacks by Manduca sexta larvae. The similarities between the defense signaling pathway in tomato leaves and those of the defense signaling pathways of macrophages and mast cells of animals suggests that both the plant and animal pathways may have evolved from a common ancestral origin.
Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.
Authors: Nolta J A , Dao M A , Wells S , Smogorzewska E M , Kohn D B
Published: July 2002

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XIS) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced.
Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis.
Authors: Wang Q , Stacy T , Binder M , Marin-Padilla M , Sharpe A H , Speck N A
Published: July 2002

The CBFA2 (AML1) gene encodes a DNA-binding subunit of the heterodimeric core-binding factor. The CBFA2 gene is disrupted by the (8;21), (3;21), and (12;21) chromosomal translocations associated with leukemias and myelodysplasias in humans. Mice lacking a CBF alpha 2 protein capable of binding DNA die between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system (CNS), at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging is preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis do not occur in Cbfa2-deficient embryos, and disruption of one copy of the Cbfa2 gene significantly reduces the number of progenitors for erythroid and myeloid cells.
Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts.
Authors: Lewis W , Levine E S , Griniuviene B , Tankersley K O , Colacino J M , Sommadossi J P , Watanabe K A , Perrino F W
Published: July 2002

The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.
Vaccination against colonizing bacteria with multiple serotypes
Authors: Lipsitch Marc
Published: July 2002

Conjugate vaccines protect vaccinated individuals against both disease from and nasopharyngeal carriage ofStreptococcus pneumoniaeandHaemophilus influenzae. Protection is specific to the capsular serotype(s) included in the vaccine. This specificity has raised concern that vaccination against particular (“targeted”) serotypes may cause an increase in carriage of (and diseases attributable to) nontargeted serotypes. I analyzed a mathematical model designed to predict the factors affecting, and the expected extent of, such replacement in the host population. The conditions for competitive exclusion and coexistence of serotypes under mass vaccination are derived, and the equilibrium carriage of target and nontarget serotypes is determined under various ecological and epidemiological conditions. The eradication threshold for a target serotype in the presence of competing, nontarget serotypes is always lower for serotype-specific than for bivalent vaccines. In a two-serotype model, the increase in the prevalence of any single nontargeted serotype due to vaccination will not exceed the total reduction in prevalence of a targeted serotype. However, if three or more serotypes interact epidemiologically, vaccination against one type may increase carriage of a second more than it decreases carriage of the first. Carriage of a second serotype against which the vaccine offers only partial protection may initially increase and then decrease as a function of vaccine coverage. I discuss the extent to which these theoretical results can account for existing data on serotype replacement after vaccination againstH. influenzaeand their implications for vaccine policy.
Identification of a nuclear matrix targeting signal in the leukemia and bone-related AML/CBF-α transcription factors
Authors: Zeng Congmei , van Wijnen André J. , Stein Janet L. , Meyers Shari , Sun Wuhua , Shopland Lindsay , Lawrence Jeanne B. , Penman Sheldon , Lian Jane B. , Stein Gary S. , Hiebert Scott W.
Published: July 2002

Transcription factors of the AML (core binding factor-α/polyoma enhancer binding protein 2) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. Alternative splicing of the
AML-1
gene results in two major AML variants, AML-1 and AML-1B. We show here that the transcriptionally active AML-1B binds to the nuclear matrix, and the inactive AML-1 does not. The association of AML-1B with the nuclear matrix is independent of DNA binding and requires a nuclear matrix targeting signal (NMTS), a 31 amino acid segment near the C terminus that is distinct from nuclear localization signals. A similar NMTS is present in AML-2 and the bone-related AML-3 transcription factors. Fusion of the AML-1B NMTS to the heterologous GAL4-(1–147) protein directs GAL4 to the nuclear matrix. Thus, the NMTS is necessary and sufficient to target the transcriptionally active AML-1B to the nuclear matrix. The loss of the C-terminal domain of AML-1B is a frequent consequence of the leukemia-related t(8;21) and t(3;21) translocations. Our results suggest this loss may be functionally linked to the modified interrelationships between nuclear structure and gene expression characteristic of cancer cells.
Suspensor-derived polyembryony caused by altered expression of valyl-tRNA synthetase in the
twn2
mutant of
Arabidopsis
Authors: Zhang James Z. , Somerville Chris R.
Published: July 2002

The
twn2
mutant of
Arabidopsis
exhibits a defect in early embryogenesis where, following one or two divisions of the zygote, the decendents of the apical cell arrest. The basal cells that normally give rise to the suspensor proliferate abnormally, giving rise to multiple embryos. A high proportion of the seeds fail to develop viable embryos, and those that do, contain a high proportion of partially or completely duplicated embryos. The adult plants are smaller and less vigorous than the wild type and have a severely stunted root. The
twn2-1
mutation, which is the only known allele, was caused by a T-DNA insertion in the 5′ untranslated region of a putative valyl-tRNA synthetase gene,
valRS
. The insertion causes reduced transcription of the
valRS
gene in reproductive tissues and developing seeds but increased expression in leaves. Analysis of transcript initiation sites and the expression of promoter–reporter fusions in transgenic plants indicated that enhancer elements inside the first two introns interact with the border of the T-DNA to cause the altered pattern of expression of the
valRS
gene in the
twn2
mutant. The phenotypic consequences of this unique mutation are interpreted in the context of a model, suggested by Vernon and Meinke [Vernon, D. M. & Meinke, D. W. (1994)
Dev. Biol.
165, 566–573], in which the apical cell and its decendents normally suppress the embryogenic potential of the basal cell and its decendents during early embryo development.
Protection against immunopathological consequences of a viral infection by activated but not resting cytotoxic T cells: T cell memory without “memory T cells”?
Authors: Bachmann Martin F. , Kündig Thomas M. , Hengartner Hans , Zinkernagel Rolf M.
Published: July 2002

Immunological memory is a key characteristic of specific immune responses. Persistence of increased levels of precursor T cells is antigen-independent and is often used as an indicator of T cell memory. This study documents that, depending on the chosen readout, cytotoxic T lymphocyte (CTL) memory against lymphocytic choriomeningitis virus (LCMV) appears long- or short-lived in the absence of persisting antigen. To study T cell memory in the absence of persisting antigen, either short-lived antigens were used for immunization or adoptive transfer methods were used to eliminate possibly persisting antigen. These experiments revealed that increased specific precursor frequencies and CTL-mediated protection against an i.v. infection with LCMV were long-lived. In contrast, CTL-mediated protection against a peripheral infection of the skin with LCMV, or of the ovary with recombinant vaccinia virus, was short-lived. These results show that maintenance of increased specific CTL precursor frequencies and central T cell memory in lymphoid tissue (where preexisting neutralizing antibodies usually provide protection anyway) is long-lived and antigen-independent. In contrast, in protection against peripheral viral infections, where the relative kinetics of virus growth and virus elimination by T cells are of key importance, T cell memory is short-lived in the absence of antigen. This indicates that peripheral T cell memory in antibody-inaccessible tissues is mediated by antigen-activated effector T cells and apparently not by specialized memory T cells.
Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation
Authors: Borhani David W. , Rogers Danise P. , Engler Jeffrey A. , Brouillette Christie G.
Published: July 2002

The structure of truncated human apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein, has been determined at 4-Å resolution. The crystals comprise residues 44–243 (exon 4) of apo A-I, a fragment that binds to lipid similarly to intact apo A-I and that retains the lipid-bound conformation even in the absence of lipid. The molecule consists almost entirely of a pseudo-continuous, amphipathic α-helix that is punctuated by kinks at regularly spaced proline residues; it adopts a shape similar to a horseshoe of dimensions 125 × 80 × 40 Å. Four molecules in the asymmetric unit associate via their hydrophobic faces to form an antiparallel four-helix bundle with an elliptical ring shape. Based on this structure, we propose a model for the structure of apo A-I bound to high density lipoprotein.
Identification of semaphorin E as a non-MDR drug resistance gene of human cancers
Authors: Yamada Tesshi , Endo Ritsuko , Gotoh Masahiro , Hirohashi Setsuo
Published: July 2002

To improve cancer chemotherapy, a better understanding of the molecular mechanisms of drug resistance is essential. To identify the molecules responsible for drug resistance that is unrelated to
MDR1
or
MRP
gene products, a eukaryotic expression cDNA library of
cis
-diamminedichloroplatinum(II) (CDDP)-resistant ovarian cancer TYKnuR cells was introduced into Cos-7 cells. After repeated CDDP selection, cDNA homologous to murine semaphorin E was isolated from surviving cells. Human semaphorin E (H-sema E) was overexpressed in CDDP-resistant cell lines and was readily induced not only by diverse chemotherapeutic drugs but also by x-ray and UV irradiation. Transfection of H-sema E conferred a drug-resistant phenotype to CDDP-sensitive cells. In addition, the aberrant expression of H-sema E protein was detected immunohistochemically in 14 of 42 (33.3%) recurrent squamous cell carcinomas removed at autopsy after extensive radiochemotherapy. Recently, another member of the semaphorin family, CD100, was shown to significantly improve the viability of B lymphocytes. These results suggest the involvement of semaphorins in diverse cell survival mechanisms.
The crystal structure of subtilisin Carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile
Authors: Schmitke Jennifer L. , Stern Lawrence J. , Klibanov Alexander M.
Published: July 2002

The x-ray crystal structure of the serine protease subtilisin Carlsberg in anhydrous dioxane has been determined to 2.6-Å resolution. The enzyme structure is found to be nearly indistinguishable from the structures previously determined in water and acetonitrile. Small changes in the side-chain conformations between the dioxane and water structures are of the same magnitude as those observed between two structures in different aqueous systems. Seven enzyme-bound dioxane molecules have been detected, each potentially forming at least one hydrogen bond with a subtilisin hydrogen-bond donor or bound water. Two of the bound dioxane molecules are in the active-site region, one in the P2 and another bridging the P1′ and P3′ pockets. The other five dioxane molecules are located on the surface of subtilisin at interprotein crystal contacts. The locations of the bound solvent in the dioxane structure are distinct from those in the structures in acetonitrile and in water.
Pancreatic β cells synthesize and secrete nerve growth factor
Authors: Rosenbaum Tamara , Vidaltamayo Román , Sánchez-Soto Ma. Carmen , Zentella Alejandro , Hiriart Marcia
Published: July 2002

Differentiation and function of pancreatic β cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic β cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in β cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic β-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.
Short-term regulation of insulin gene transcription by glucose
Authors: Leibiger Barbara , Moede Tilo , Schwarz Thomas , Brown Graham R. , Köhler Martin , Leibiger Ingo B. , Berggren Per-Olof
Published: July 2002

Whereas short-term regulation of insulin biosynthesis at the level of translation is well accepted, glucose-dependent transcriptional control is still believed to be a long-term effect occurring after more than 2 hr of glucose stimulation. Because pancreatic β cells are exposed to elevated glucose levels for minutes rather than hours after food uptake, we hypothesized the existence of a short-term transcriptional control. By studying the dynamics of newly synthesized (prepro)insulin RNA and by employing on-line monitoring of gene expression in single, insulin-producing cells, we were able to provide convincing evidence that insulin gene transcription indeed is affected by glucose within minutes. Exposure of insulinoma cells and isolated pancreatic islets to elevated glucose for only 15 min resulted in a 2- to 5-fold elevation in (prepro)insulin mRNA levels within 60–90 min. Similarly, insulin promoter-driven green fluorescent protein expression in single insulin-producing cells was significantly enhanced after transient glucose stimulation. Thus, short-term signaling, such as that involved in insulin secretion, also may regulate insulin gene transcription.