bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary bioflavonoids induce cleavage in the
MLL
gene and may contribute to infant leukemia
Authors: Strick Reiner , Strissel Pamela L. , Borgers Susanne , Smith Steve L. , Rowley Janet D.
Published: July 2002

Chromosomal translocations involving the
MLL
gene occur in about 80% of infant leukemia. In the search for possible agents inducing infant leukemia, we identified bioflavonoids, natural substances in food as well as in dietary supplements, that cause site-specific DNA cleavage in the
MLL
breakpoint cluster region (BCR)
in vivo
. The
MLL
BCR DNA cleavage was shown in primary progenitor hematopoietic cells from healthy newborns and adults as well as in cell lines; it colocalized with the
MLL
BCR cleavage site induced by chemotherapeutic agents, such as etoposide (VP16) and doxorubicin (Dox). Both
in vivo
and additional
in vitro
experiments demonstrated topoisomerase II (topo II) as the target of bioflavonoids similar to VP16 and Dox. Based on 20 bioflavonoids tested, we identified a common structure essential for topo II-induced DNA cleavage. Reversibility experiments demonstrated a religation of the bioflavonoid as well as the VP16-induced
MLL
cleavage site. Our observations support a two-stage model of cellular processing of topo II inhibitors: The first and reversible stage of topo II-induced DNA cleavage results in DNA repair, but also rarely in chromosome translocations; whereas the second, nonreversible stage leads to cell death because of an accumulation of DNA damage. These results suggest that maternal ingestion of bioflavonoids may induce
MLL
breaks and potentially translocations
in utero
leading to infant and early childhood leukemia.
RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: A negative feedback circuit
Authors: Xie Youming , Varshavsky Alexander
Published: July 2002

The RPN4 (SON1, UFD5) protein of the yeast
Saccharomyces
cerevisiae
is required for normal levels of intracellular
proteolysis. RPN4 is a transcriptional activator of genes encoding
proteasomal subunits. Here we show that RPN4 is required for normal
levels of these subunits. Further, we demonstrate that RPN4 is
extremely short-lived (
t
1/2
≈2 min), that
it directly interacts with RPN2, a subunit of the 26S proteasome, and
that
rpn4Δ
cells are perturbed in their cell cycle.
The degradation signal of RPN4 was mapped to its N-terminal region,
outside the transcription–activation domains of RPN4. The ability of
RPN4 to augment the synthesis of proteasomal subunits while being
metabolically unstable yields a negative feedback circuit in which the
same protein up-regulates the proteasome production and is destroyed by
the assembled active proteasome.
The
in vitro
and
in vivo
phosphotyrosine map of activated MuSK
Authors: Watty Anke , Neubauer Gitte , Dreger Mathias , Zimmer Manuel , Wilm Matthias , Burden Steven J.
Published: July 2002

The muscle-specific receptor tyrosine kinase MuSK plays a crucial role in neuromuscular synapse formation. Activation of MuSK is induced by agrin leading to clustering of several proteins, including acetylcholine receptors, at synaptic sites. In a first step to elucidate the signal transduction cascade following MuSK activation and leading to clustering of synaptic proteins, we sought to identify the tyrosine residues that are phosphorylated in activated MuSK. We mapped the tyrosine residues that are phosphorylated
in vitro
and
in vivo
using methods that provide high sensitivity and do not require radioactive tracers. We expressed MuSK in insect cells by using a baculovirus expression vector and mapped the tyrosines that are phosphorylated in MuSK in an
in vitro
kinase assay using matrix-assisted laser desorption ionization MS to sequence tryptic peptides fractionated by HPLC. In addition, we isolated MuSK from
Torpedo
electric organ and used nanoelectrospray tandem mass spectrometry and parent ion scanning to identify the tyrosine residues that are phosphorylated in activated, endogenous MuSK
in vivo
. We found that six of the nineteen intracellular tyrosine residues in MuSK are phosphorylated in activated MuSK: the juxtamembrane tyrosine (Y553), the tyrosines within the activation loop (Y750, Y754, and Y755), a tyrosine near the beginning of the kinase domain (Y576), and a tyrosine (Y812) within the C-terminal lobe of the kinase domain. Our biochemical data are consistent with results from functional experiments and establish a good correlation between tyrosine residues that are phosphorylated in activated MuSK and tyrosines that are required for MuSK signaling.
Signal-dependent activation of the MEF2 transcription factor by dissociation from histone deacetylases
Authors: Lu Jianrong , McKinsey Timothy A. , Nicol Rebekka L. , Olson Eric N.
Published: July 2002

Myocyte enhancer factor-2 (MEF2) transcription factors control muscle-specific and growth factor-inducible genes. We show that hypertrophic growth of cardiomyocytes in response to phenylephrine and serum is accompanied by activation of MEF2 through a posttranslational mechanism mediated by calcium, calmodulin-dependent protein kinase (CaMK), and mitogen-activated protein kinase (MAPK) signaling. CaMK stimulates MEF2 activity by dissociating class II histone deacetylases (HDACs) from the DNA-binding domain. MAPKs, which activate MEF2 by phosphorylation of the transcription activation domain, maximally stimulate MEF2 activity only when repression by HDACs is relieved by CaMK signaling to the DNA-binding domain. These findings identify MEF2 as an endpoint for hypertrophic stimuli in cardiomyocytes and demonstrate that MEF2 mediates synergistic transcriptional responses to the CaMK and MAPK signaling pathways by signal-dependent dissociation from HDACs.
Myc represses the p21
(WAF1/CIP1)
promoter and interacts with Sp1/Sp3
Authors: Gartel Andrei L. , Ye Xin , Goufman Eugene , Shianov Pavel , Hay Nissim , Najmabadi Feridoon , Tyner Angela L.
Published: July 2002

The cyclin-dependent kinase inhibitor p21
(WAF1/CIP1)
inhibits proliferation both
in vitro
and
in
vivo
, and overexpression of p21 in normal and tumor cell lines
results in cell cycle arrest. In contrast, ectopic expression of Myc
alleviates G
1
cell cycle arrest. Recent studies showed that
Myc can repress p21 transcription, thereby overriding a p21-mediated
cell cycle checkpoint. We found that activation of a Myc-estrogen
receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted
in suppression of endogenous p21 transcription. This effect
was observed in the absence of
de novo
protein synthesis
and was independent of histone deacetylase activity. In transient
transfection studies, Myc effectively repressed p21 promoter constructs
containing only 119 bp of sequence upstream of the transcription start
site. This region contains multiple Sp1-binding sites and a potential
initiator element, but no canonical Myc DNA-binding sites. Deletion of
the potential initiator element does not affect repression of the p21
promoter by c-Myc. Coimmunoprecipitation and glutathione
S
-transferase pull-down experiments demonstrate that
c-Myc may form complexes with Sp1/Sp3. We found that the central
region of c-Myc interacts with the zinc finger domain of Sp1. Because
Sp1 is required for p21 transcription, it is possible that Myc may
down-regulate p21 transcription, at least in part, by sequestering Sp1.
Repression of the p21 promoter may contribute to the ability of c-Myc
to promote cell proliferation.
Characterization of the c-MYC-regulated transcriptome by SAGE: Identification and analysis of c-MYC target genes
Authors: Menssen Antje , Hermeking Heiko
Published: July 2002

To identify target genes of the oncogenic transcription factor c-MYC, serial analysis of gene expression (SAGE) was performed after adenoviral expression of c-
MYC
in primary human umbilical vein endothelial cells: 216 different SAGE tags, corresponding to unique mRNAs, were induced, whereas 260 tags were repressed after c-MYC expression (
P
< 0.05). The induction of 53 genes was confirmed by using microarray analysis and quantitative real-time PCR: among these genes was
MetAP2
/
p67
, which encodes an activator of translational initiation and represents a validated target for inhibition of neovascularization. Furthermore, c-MYC induced the cell cycle regulatory genes
CDC2-L1
,
Cyclin E binding protein 1
, and
Cyclin B1
. The DNA repair genes
BRCA1
,
MSH2
, and
APEX
were induced by c-MYC, suggesting that c-
MYC
couples DNA replication to processes preserving the integrity of the genome.
MNT
, a MAX-binding antagonist of c-MYC function, was up-regulated, implying a negative feedback loop.
In vivo
promoter occupancy by c-MYC was detected by chromatin immunoprecipitation for
CDK4
,
Prohibitin
,
MNT
,
Cyclin B1
, and
Cyclin E binding protein 1
, showing that these genes are direct c-MYC targets. The c-MYC-regulated genes/tags identified here will help to define the set of bona fide c-MYC targets and may have potential therapeutic value for inhibition of cancer cell proliferation, tumor-vascularization, and restenosis.
Altered sexual and social behaviors in trp2 mutant mice
Authors: Leypold Bradley G. , Yu C. Ron , Leinders-Zufall Trese , Kim Michelle M. , Zufall Frank , Axel Richard
Published: July 2002

We have used gene targeting to generate mice with a homozygous deficiency in trp2, a cation channel expressed in the vomeronasal organ (VNO). Trp2 mutant animals reveal a striking reduction in the electrophysiological response to pheromones in the VNO, suggesting that trp2 plays a central role in mediating the pheromone response. These mutants therefore afford the opportunity to examine the role of the VNO in the generation of innate sexual and social behaviors in mice. Trp2 mutant males and nursing females are docile and fail to initiate aggressive attacks on intruder males. Male–female sexual behavior appears normal, but trp2 mutant males also vigorously mount other males. These results suggest that the cation channel trp2 is required in the VNO to detect male-specific pheromones that elicit aggressive behaviors and dictate the choice of sexual partners.
YgbQ, a cell division protein in
Escherichia coli
and
Vibrio cholerae
, localizes in codependent fashion with FtsL to the division site
Authors: Buddelmeijer Nienke , Judson Nicholas , Boyd Dana , Mekalanos John J. , Beckwith Jonathan
Published: July 2002

YgbQ is a cell division protein in
Escherichia coli
and
Vibrio cholerae
. In
E. coli
the
ygbQ
gene was discovered as a result of a computer search of the
E. coli
genome designed to find potential interacting partners for cell division protein FtsL. In
V. cholerae
,
ygbQ
was identified as an essential gene by using a transposon that fuses genes to an arabinose promoter. The role of YgbQ in cell division is supported by the following. Cells depleted of YgbQ in both organisms form long filaments, but DNA segregation is not affected. YgbQ localizes to the constriction site in wild-type
E. coli
cells. Localization of
E. coli
YgbQ to the constriction site depends on cell division proteins FtsQ and FtsL but not FtsW and FtsI, placing YgbQ in the sequential dependency order of proteins localizing to the division site. Localization of green fluorescent protein-FtsL also depends on YgbQ, indicating that FtsL and YgbQ colocalize to the division site in
E. coli
. Our results show colocalization of proteins to the bacterial midcell in
E. coli
and raise the possibility that these proteins interact in a coiled-coil structure.
Centripetal and centrifugal reorganizations of frequency map of auditory cortex in gerbils
Authors: Sakai Masashi , Suga Nobuo
Published: July 2002

As repetitive acoustic stimulation and auditory conditioning do, electric stimulation of the primary auditory cortex (AI) evokes reorganization of the frequency map of AI, as well as of the subcortical auditory nuclei. The reorganization is caused by shifts in best frequencies (BFs) of neurons either toward (centripetal) or away from (centrifugal) the BF of stimulated cortical neurons. In AI of the Mongolian gerbil, we found that focal electrical stimulation evoked a centripetal BF shift in an elliptical area centered at the stimulated neurons and a centrifugal BF shift in a zone surrounding it. The 1.9-mm long major and 1.1-mm long minor axes of the elliptical area were parallel and orthogonal to the frequency axis, respectively. The width of the surrounding zone was 0.2–0.3 mm. Such “center-surround” reorganization has not yet been found in any sensory cortex except AI of the gerbil. The ellipse is similar to the arborization pattern of pyramidal neurons, the major source of excitatory horizontal connections in AI, whereas the surrounding zone is compatible to the arborization range of small basket cells (inhibitory neurons) in AI.
Variola virus immune evasion design: Expression of a highly efficient inhibitor of human complement
Authors: Rosengard Ariella M. , Liu Yu , Nie Zhiping , Jimenez Robert
Published: July 2002

Variola virus, the most virulent member of the genusOrthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30–40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges.
Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (
Arnt2
) gene reveals partial redundancy with
Arnt
Authors: Keith Brian , Adelman David M. , Simon M. Celeste
Published: July 2002

The ubiquitously expressed basic helix–loop–helix (bHLH)-PAS
protein ARNT (
a
rylhydrocarbon
r
eceptor
n
uclear
t
ransporter) forms transcriptionally
active heterodimers with a variety of other bHLH-PAS proteins,
including HIF-1α (
h
ypoxia-
i
nducible
f
actor-1α) and AHR
(
a
ryl
h
ydrocarbon
r
eceptor). These
complexes regulate gene expression in response to hypoxia and
xenobiotics, respectively, and mutation of the murine
Arnt
locus results in embryonic death by day 10.5
associated with placental, vascular, and hematopoietic defects. The
closely related protein ARNT2 is highly expressed in the central
nervous system and kidney and also forms complexes with HIF-1α and
AHR. To assess unique roles for ARNT2 in development, and reveal
potential functional overlap with ARNT, we generated a targeted null
mutation of the murine
Arnt2
locus.
Arnt2
−/−
embryos die perinatally and
exhibit impaired hypothalamic development, phenotypes previously
observed for a targeted mutation in the murine bHLH-PAS gene
Sim1
(Single-minded 1), and consistent with the recent
proposal that ARNT2 and SIM1 form an essential heterodimer
in
vivo
[Michaud, J. L., DeRossi, C., May, N. R.,
Holdener, B. C. & Fan, C. (2000)
Mech. Dev.
90,
253–261]. In addition, cultured
Arnt2
−/−
neurons display decreased hypoxic induction of HIF-1 target genes,
demonstrating formally that ARNT2/HIF-1α complexes regulate
oxygen-responsive genes. Finally, a strong genetic interaction between
Arnt
and
Arnt2
mutations was observed,
indicating that either gene can fulfill essential functions in a
dose-dependent manner before embryonic day 8.5. These results
demonstrate that
Arnt
and
Arnt2
have both
unique and overlapping essential functions in embryonic development.
The transcriptional corepressor MITR is a signal-responsive inhibitor of myogenesis
Authors: Zhang Chun Li , McKinsey Timothy A. , Olson Eric N.
Published: July 2002

Activation of muscle-specific genes by members of the myocyte enhancer factor 2 (MEF2) and MyoD families of transcription factors is coupled to histone acetylation and is inhibited by class II histone deacetylases (HDACs) 4 and 5, which interact with MEF2. The ability of HDAC4 and -5 to inhibit MEF2 is blocked by phosphorylation of these HDACs at two conserved serine residues, which creates docking sites for the intracellular chaperone protein 14-3-3. When bound to 14-3-3, HDACs are released from MEF2 and transported to the cytoplasm, thereby allowing MEF2 to stimulate muscle-specific gene expression. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal regions of HDAC4 and -5, but lacks an HDAC catalytic domain. Despite the absence of intrinsic HDAC activity, MITR acts as a potent inhibitor of MEF2-dependent transcription. Paradoxically, however, MITR has minimal inhibitory effects on the skeletal muscle differentiation program. We show that a substitution mutant of MITR containing alanine in place of two serine residues, Ser-218 and Ser-448, acts as a potent repressor of myogenesis. Our findings indicate that promyogenic signals antagonize the inhibitory action of MITR by targeting these serines for phosphorylation. Phosphorylation of Ser-218 and Ser-448 stimulates binding of 14-3-3 to MITR, disrupts MEF2:MITR interactions, and alters the nuclear distribution of MITR. These results reveal a role for MITR as a signal-dependent regulator of muscle differentiation.
A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops
Authors: Tomita Kozo , Ogawa Tetsuhiro , Uozumi Takeshi , Watanabe Kimitsuna , Masaki Haruhiko
Published: July 2002

Colicin D has long been thought to stop protein synthesis in infected
Escherichia coli
cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAs
Arg
including four isoaccepting molecules both
in vivo
and
in vitro
. The cleavage occurs
in vitro
between positions 38 and 39 in an anticodon loop with a 2′,3′-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAs
Arg
, the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity
in vivo
together with the tRNA
Arg
-cleaving activity both
in vivo
and
in vitro
. These findings show that colicin D directly cleaves cytoplasmic tRNAs
Arg
, which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.
C/EBPɛ mediates myeloid differentiation and is regulated by the CCAAT displacement protein (CDP/cut)
Authors: Khanna-Gupta Arati , Zibello Theresa , Sun Hong , Lekstrom-Himes Julie , Berliner Nancy
Published: July 2002

Neutrophils from CCAAT enhancer binding protein epsilon (C/EBPɛ) knockout mice have morphological and biochemical features similar to those observed in patients with an extremely rare congenital disorder called neutrophil-specific secondary granule deficiency (SGD). SGD is characterized by frequent bacterial infections attributed, in part, to the lack of neutrophil secondary granule proteins (SGP). A mutation that results in loss of functional C/EBPɛ activity has recently been described in an SGD patient, and has been postulated to be the cause of the disease in this patient. We have previously demonstrated that overexpression of CCAAT displacement protein (CDP/cut), a highly conserved transcriptional repressor of developmentally regulated genes, suppresses expression of SGP genes in 32Dcl3 cells. This phenotype resembles that observed in both C/EBPɛ−/− mice and in SGD patients. Based on these observations we investigated potential interactions between C/EBPɛ and CDP/cut during neutrophil maturation. In this study, we demonstrate that inducible expression of C/EBPɛ in 32Dcl3/tet cells results in granulocytic differentiation. Furthermore, Northern blot analysis of G-CSF-induced CDP/cut overexpressing 32Dcl3 cells revealed absence of C/EBPɛ mRNA. We therefore hypothesize that C/EBPɛ positively regulates SGP gene expression, and that C/EBPɛ is itself negatively regulated by CDP/cut during neutrophil maturation. We further demonstrate that the C/EBPɛ promoter is regulated by CDP/cut during myeloid differentiation.
Cortical processing of change detection: Dissociation between natural vowels and two-frequency complex tones
Authors: Vihla Minna , Lounasmaa Olli V. , Salmelin Riitta
Published: July 2002

We compared magnetoencephalographic responses for natural vowels and for sounds consisting of two pure tones that represent the two lowest formant frequencies of these vowels. Our aim was to determine whether spectral changes in successive stimuli are detected differently for speech and nonspeech sounds. The stimuli were presented in four blocks applying an oddball paradigm (20% deviants, 80% standards): (
i
) /α/ tokens as deviants vs. /i/ tokens as standards; (
ii
) /e/ vs. /i/; (
iii
) complex tones representing /α/ formants vs. /i/ formants; and (
iv
) complex tones representing /e/ formants vs. /i/ formants. Mismatch fields (MMFs) were calculated by subtracting the source waveform produced by standards from that produced by deviants. As expected, MMF amplitudes for the complex tones reflected acoustic deviation: the amplitudes were stronger for the complex tones representing /α/ than /e/ formants, i.e., when the spectral difference between standards and deviants was larger. In contrast, MMF amplitudes for the vowels were similar despite their different spectral composition, whereas the MMF onset time was longer for /e/ than for /α/. Thus the degree of spectral difference between standards and deviants was reflected by the MMF amplitude for the nonspeech sounds and by the MMF latency for the vowels.
Specificity in intracellular protein aggregation and inclusion body formation
Authors: Rajan Rahul S. , Illing Michelle E. , Bence Neil F. , Kopito Ron R.
Published: July 2002

Protein aggregation is widely considered to be a nonspecific coalescence of misfolded proteins, driven by interactions between solvent-exposed hydrophobic surfaces that are normally buried within a protein's interior. Accordingly, abnormal interactions between misfolded proteins with normal cellular constituents has been proposed to underlie the toxicity associated with protein aggregates in many neurodegenerative disorders. Here we have used fluorescence resonance energy transfer and deconvolution microscopy to investigate the degree to which unrelated misfolded proteins expressed in the same cells coaggregate with one another. Our data reveal that in cells, protein aggregation exhibits exquisite specificity even among extremely hydrophobic substrates expressed at very high levels.
Ricin A chain without its partner B chain is degraded after retrotranslocation from the endoplasmic reticulum to the cytosol in plant cells
Authors: Di Cola Alessandra , Frigerio Lorenzo , Lord J. Michael , Ceriotti Aldo , Roberts Lynne M.
Published: July 2002

When expressed in tobacco cells, the catalytic subunit of the
dimeric ribosome inactivating protein, ricin, is first inserted into
the endoplasmic reticulum (ER) and then degraded in a manner that can
be partially inhibited by the proteasome inhibitor
clasto
-lactacystin β-lactone. Consistent with
the implication of cytosolic proteasomes, degradation of ricin A chain
is brefeldin A-insensitive and the polypeptides that accumulate in the
presence of the proteasome inhibitor are not processed in a
vacuole-specific fashion. Rather, these stabilized polypeptides are in
part deglycosylated by a peptide:
N
-glycanase-like
activity. Taken together, these results indicate that ricin A chain,
albeit a structurally native protein, can behave as a substrate for ER
to cytosol export, deglycosylation in the cytosol, and proteasomal
degradation. Furthermore, retrotranslocation of this protein is not
tightly coupled to proteasomal activity. These data are consistent with
the hypothesis that ricin A chain can exploit the ER-associated protein
degradation pathway to reach the cytosol. Although well
characterized in mammalian and yeast cells, the operation of a similar
pathway to the cytosol of plant cells has not previously been
demonstrated.
A high level of cyclooxygenase-2 inhibitor selectivity is associated with a reduced interference of platelet cyclooxygenase-1 inactivation by aspirin
Authors: Ouellet Marc , Riendeau Denis , Percival M. David
Published: July 2002

Both nonsteroidal anti-inflammatory drugs, such as ibuprofen, and
the prototypical selective cyclooxygenase (Cox)-2 inhibitors DuP-697
and NS-398 block the inhibition of Cox-1 by aspirin
in
vitro
. However, clinical studies have shown that the
Cox-2 selective drugs (or coxibs) rofecoxib and etoricoxib, at
therapeutic doses, do not interfere with the antiplatelet effect of
aspirin, in contrast to ibuprofen. Here, we have evaluated the relative
potential of ibuprofen and various coxibs to interfere with the
inactivation of Cox-1 by aspirin by using purified enzyme and calcium
ionophore-activated human platelets. The irreversible inactivation of
Cox-1 by aspirin can be antagonized by ibuprofen and coxibs, albeit
with widely different potencies. The rank order of potencies for this
process (ibuprofen > celecoxib > valdecoxib >
rofecoxib > etoricoxib) parallels that obtained for the
inhibition of Cox-1-mediated thromboxane B
2
production by
calcium ionophore-stimulated platelets. The antagonism of aspirin
therefore likely involves a competition at the enzyme active site. The
EC
50
value for the antagonism against 10 μM aspirin for
each drug is ≈10- to 40-fold lower than the corresponding
IC
50
value for inhibition of platelet Cox-1 activity,
consistent with the much weaker initial binding of aspirin to Cox-1 as
compared with arachidonic acid. These results show that a low affinity
for Cox-1 and a high degree of Cox-2 selectivity confers a low
potential to block aspirin inhibition of platelet Cox-1, consistent
with the results of clinical studies.
Suppression of proliferation and cardiomyocyte hypertrophy by CHAMP, a cardiac-specific RNA helicase
Authors: Liu Zhi-Ping , Olson Eric N.
Published: July 2002

Adult cardiomyocytes are irreversibly postmitotic but respond to a variety of stimuli by hypertrophic growth, which is associated with an increase in cell size and protein content, organization of sarcomeres, and activation of a fetal gene program. Recently, we described a novel cardiac helicase activated by MEF2 protein (CHAMP), which is expressed specifically in the heart throughout prenatal and postnatal development. Here we show that CHAMP acts as an inhibitor of cell proliferation and cardiomyocyte hypertrophy. Ectopic expression of CHAMP inhibits proliferation of HeLa cells and blocks cell cycle entry of serum-stimulated NIH 3T3 cells. Overexpression of CHAMP in primary neonatal cardiomyocytes blocks hypertrophic growth and the induction of fetal genes in response to stimulation by serum and phenylephrine but does not prevent sarcomere organization or early mitogenic signaling events including activation of extracellular signal-regulated kinases or up-regulation of
c-fos
. Inhibition of cardiomyocyte hypertrophy by CHAMP requires the conserved ATPase domain and is accompanied by up-regulation of the cyclin-dependent protein kinase inhibitor p21
CIP1
. These findings identify CHAMP as a cardiac-specific suppressor of cardiomyocyte hypertrophy and cell cycle progression and suggest that CHAMP may suppress these processes through the regulation of p21
CIP1
.
Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis.
Authors: Abeijon C , Robbins P W , Hirschberg C B
Published: July 2002

The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha1-->2-linked N-acetylglucosamine. The biosynthesis of these chains probably occurs in the lumen of the Golgi apparatus, by analogy to S. cerevisiae. The sugar donors, GDP-mannose and UDP-GlcNAc, must first be transported from the cytosol, their site of synthesis, via specific Golgi membrane transporters into the lumen where they are substrates in the biosynthesis of these mannoproteins. A mutant of K. lactis, mnn2-2, that lacks terminal N-acetylglucosamine in its mannan chains in vivo, has recently been characterized and shown to have a specific defect in transport of UDP-GlcNAc into the lumen of Golgi vesicles in vitro. We have now cloned the gene encoding the K. lactis Golgi membrane UDP-GlcNAc transporter by complementation of the mnn2-2 mutation. The mnn2-2 mutant was transformed with a genomic library from wild-type K. lactis in a pKD1-derived vector; transformants were isolated and phenotypic correction was monitored following cell surface labeling with fluorescein isothiocyanate conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescent activated cell sorter. A 2.4-kb DNA fragment was found to restore the wild-type lectin binding phenotype. Upon loss of the plasmid containing this fragment, reversion to the mutant phenotype occurred. The above fragment contained an open reading frame for a multitransmembrane spanning protein of 328 amino acids. The protein contains a leucine zipper motif and has high homology to predicted proteins from S. cerevisiae and C. elegans. In an assay in vitro, Golgi vesicles isolated from the transformant had regained their ability to transport UDP-GlcNAc. Taken together, the above results strongly suggest that the cloned gene encodes the Golgi UDP-GlcNAc transporter of K. lactis.
An axoplasmic myosin with a calmodulin-like light chain.
Authors: Bearer E L , DeGiorgis J A , Jaffe H , Medeiros N A , Reese T S
Published: July 2002

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.
Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication.
Authors: Malkova A , Ivanov E L , Haber J E
Published: July 2002

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.
Expression cloning of the Golgi CMP-sialic acid transporter.
Authors: Eckhardt M , Mühlenhoff M , Bethe A , Gerardy-Schahn R
Published: July 2002

Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.
An RNA topoisomerase.
Authors: Wang H , Di Gate R J , Seeman N C
Published: July 2002

A synthetic strand of RNA has been designed so that it can adopt two different topological states (a circle and a trefoil knot) when ligated into a cyclic molecule. The RNA knot and circle have been characterized by their behavior in gel electrophoresis and sedimentation experiments. This system allows one to assay for the existence of an RNA topoisomerase, because the two RNA molecules can be inter-converted only by a strand passage event. We find that the interconversion of these two species can be catalyzed by Escherichia coli DNA topoisomerase III, indicating that this enzyme can act as an RNA topoisomerase. The conversion of circles to knots is accompanied by a small amount of RNA catenane generation. These findings suggest that strand passage must be considered a potential component of the folding and modification of RNA structures.
Identification of potential target genes for the neuron-restrictive silencer factor.
Authors: Schoenherr C J , Paquette A J , Anderson D J
Published: July 2002

The neuron-restrictive silencer factor (NRSF) represses transcription of several neuronal genes in nonneuronal cells by binding to a 21-bp element called the neuron-restrictive silencer element (NRSE). We have performed data base searches with a composite NRSE to identify additional candidate NRSF target genes. Twenty-two more genes, 17 of which are expressed mainly in neurons, were found to contain NRSE-like sequences. Many of these putative NRSEs bound NRSF in vitro and repressed transcription in vivo. Most of the neuronal genes identified contribute to the basic structural or functional properties of neurons. However, two neuronal transcription factor genes contain NRSEs, suggesting that NRSF may repress neuronal differentiation both directly and indirectly. Functional NRSEs were also found in several nonneuronal genes, implying that NRSF may play a broader role than originally anticipated.
Protonation dynamics of the extracellular and cytoplasmic surface of bacteriorhodopsin in the purple membrane.
Authors: Nachliel E , Gutman M , Kiryati S , Dencher N A
Published: July 2002

The dynamics of proton binding to the extracellular and the cytoplasmic surfaces of the purple membrane were measured by laser-induced proton pulses. Purple membranes, selectively labeled by fluorescein at Lys-129 of bacteriorhodopsin, were pulsed by protons released in the aqueous bulk from excited pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) and the reaction of protons with the indicators was measured. Kinetic analysis of the data imply that the two faces of the membrane differ in their buffer capacities and in their rates of interaction with bulk protons. The extracellular surface of the purple membrane contains one anionic proton binding site per protein molecule with pK = 5.1. This site is within a Coulomb cage radius (approximately 15 A) from Lys-129. The cytoplasmic surface of the purple membrane bears 4-5 protonable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3.10(10) M-1.s-1). The proximity between the elements is sufficiently high that even in 100 mM NaCl they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. The protonation dynamics determined at the surface of the purple membrane is of relevance both for the vectorial proton transport mechanism of bacteriorhodopsin and for energy coupling, not only in halobacteria, but also in complex chemiosmotic systems such as mitochondrial and thylakoid membranes.
The human AQP4 gene: definition of the locus encoding two water channel polypeptides in brain.
Authors: Lu M , Lee M D , Smith B L , Jung J S , Agre P , Verdijk M A , Merkx G , Rijss J P , Deen P M
Published: July 2002

The aquaporin family of membrane water transport proteins are expressed in diverse tissues, and in brain the predominant water channel protein is AQP4. Here we report the isolation and characterization of the human AQP4 cDNAs and genomic DNA. Two cDNAs were isolated corresponding to the two initiating methionines (M1 in a 323-aa polypeptide and M23 in a 301-aa polypeptide) previously identified in rat [Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B. & Agre, P. (1994) Proc. Natl. Acad. Sci. USA 91, 13052-13056]. Similar to other aquaporins, the AQP4 gene is composed of four exons encoding 127, 55, 27, and 92 amino acids separated by introns of 0.8, 0.3, and 5.2 kb. Unlike other aquaporins, an alternative coding initiation sequence (designated exon 0) was located 2.7 kb upstream of exon 1. When spliced together, M1 and the subsequent 10 amino acids are encoded by exon 0; the next 11 amino acids and M23 are encoded by exon 1. Transcription initiation sites have been mapped in the proximal promoters of exons 0 and 1. RNase protection revealed distinct transcripts corresponding to M1 and M23 mRNAs, and AQP4 immunoblots of cerebellum demonstrated reactive polypeptides of 31 and 34 kDa. Using a P1 and a lambda EMBL subclone, the chromosomal site of the human AQP4 gene was mapped to chromosome 18 at the junction of q11.2 and q12.1 by fluorescence in situ hybridization. These studies may now permit molecular characterization of AQP4 during human development and in clinical disorders.
Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis.
Authors: Hoeg J M , Santamarina-Fojo S , Bérard A M , Cornhill J F , Herderick E E , Feldman S H , Haudenschild C C , Vaisman B L , Hoyt R F , Demosky S J , Kauffman R D , Hazel C M , Marcovina S M , Brewer H B
Published: July 2002

Lecithin:cholesterol acyltransferase (LCAT) is a key plasma enzyme in cholesterol and high density lipoprotein (HDL) metabolism. Transgenic rabbits overexpressing human LCAT had 15-fold greater plasma LCAT activity that nontransgenic control rabbits. This degree of overexpression was associated with a 6.7-fold increase in the plasma HDL cholesterol concentration in LCAT transgenic rabbits. On a 0.3% cholesterol diet, the HDL cholesterol concentrations increased from 24 +/- 1 to 39 +/- 3 mg/dl in nontransgenic control rabbits (n = 10; P < 0.05) and increased from 161 +/- 5 to 200 +/- 21 mg/dl (P < 0.001) in the LCAT transgenic rabbits (n = 9). Although the baseline non-HDL concentrations of control (4 +/- 3 mg/dl) and transgenic rabbits (18 +/- 4 mg/dl) were similar, the cholesterol-rich diet raised the non-HDL cholesterol concentrations, reflecting the atherogenic very low density, intermediate density, and low density lipoprotein particles observed by gel filtration chromatography. The non-HDL cholesterol rose to 509 +/- 57 mg/dl in controls compared with only 196 +/- 14 mg/dl in the LCAT transgenic rabbits (P < 0.005). The differences in the plasma lipoprotein response to a cholesterol-rich diet observed in the transgenic rabbits paralleled the susceptibility to developing aortic atherosclerosis. Compared with nontransgenic controls, LCAT transgenic rabbits were protected from diet-induced atherosclerosis with significant reductions determined by both quantitative planimetry (-86%; P < 0.003) and quantitative immunohistochemistry (-93%; P < 0.009). Our results establish the importance of LCAT in the metabolism of both HDL and apolipoprotein B-containing lipoprotein particles with cholesterol feeding and the response to diet-induced atherosclerosis. In addition, these findings identify LCAT as a new target for therapy to prevent atherosclerosis.
Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF.
Authors: Perkins A C , Gaensler K M , Orkin S H
Published: July 2002

Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Krüppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold. Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene.
Deglucosylation of N-linked glycans is an important step in the dissociation of calreticulin–class I–TAP complexes
Authors: Van Leeuwen Jeroen E. M. , Kearse Kelly P.
Published: July 2002

Recent evidence indicates that newly synthesized major histocompatibility complex (MHC) class I proteins interact with calnexin, a transmembrane endoplasmic reticulum protein specific for certain glycoproteins bearing monoglucosylated glycans. Here, we studied the association of newly synthesized class I proteins with calreticulin, a soluble calnexin-related ER protein, in murine T cells. We found that, unlike calnexin–class I interactions, calreticulin assembly with class I proteins was markedly decreased in the absence of β2microglobulin expression and that calreticulin associated with a subset of class I glycoforms distinct from those assembled with calnexin but similar to those bound to TAP (transporter associated with antigen processing) proteins. Finally, these studies show that deglucosylation of N-linked glycans is important for dissociation of class I proteins from both calreticulin and TAP and that the vast majority of newly synthesized class I proteins associated with calreticulin are simultaneously assembled with TAP. The data demonstrate that calnexin and calreticulin chaperones assemble with distinct MHC class I assembly intermediates in the ER and show that glycan processing is functionally coupled to release of MHC class I proteins from peptide transport molecules.
Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli.
Authors: Mellon I , Champe G N
Published: July 2002

To improve our understanding of the mechanism that couples nucleotide-excision repair to transcription in expressed genes, we have examined the effects of mutations in several different DNA repair genes on the removal of cyclobutane pyrimidine dimers from the individual strands of the induced lactose operon in UV-irradiated Escherichia coli. As expected, we found little repair in either strand of the lactose operon in strains with mutations in established nucleotide excision-repair genes (uvrA, uvrB, uvrC, or uvrD). In contrast, we found that mutations in either of two genes required for DNA-mismatch correction (mutS and mutL) selectively abolish rapid repair in the transcribed strand and render the cells moderately sensitive to UV irradiation. Similar results were found in a strain with a mutation in the mfd gene, the product of which has been previously shown to be required for transcription-coupled repair in vitro. Our results demonstrate an association between mismatch-correction and nucleotide-excision repair and implicate components of DNA-mismatch repair in transcription-coupled repair. In addition, they may have important consequences for human disease and may enhance our understanding of the etiology of certain cancers which have been associated with defects in mismatch correction.