bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene discovery and gene function assignment in filamentous fungi
Authors: Hamer Lisbeth , Adachi Kiichi , Montenegro-Chamorro Maria V. , Tanzer Matthew M. , Mahanty Sanjoy K. , Lo Clive , Tarpey Rex W. , Skalchunes Amy R. , Heiniger Ryan W. , Frank Sheryl A. , Darveaux Blaise A. , Lampe David J. , Slater Ted M. , Ramamurthy Lakshman , DeZwaan Todd M. , Nelson Grant H. , Shuster Jeffrey R. , Woessner Jeffrey , Hamer John E.
Published: July 2002

Filamentous fungi are a large group of diverse and economically
important microorganisms. Large-scale gene disruption strategies
developed in budding yeast are not applicable to these organisms
because of their larger genomes and lower rate of targeted integration
(TI) during transformation. We developed transposon-arrayed gene
knockouts (TAGKO) to discover genes and simultaneously create gene
disruption cassettes for subsequent transformation and mutant analysis.
Transposons carrying a bacterial and fungal drug resistance marker are
used to mutagenize individual cosmids or entire libraries
in
vitro
. Cosmids are annotated by DNA sequence analysis at the
transposon insertion sites, and cosmid inserts are liberated to direct
insertional mutagenesis events in the genome. Based on saturation
analysis of a cosmid insert and insertions in a fungal cosmid library,
we show that TAGKO can be used to rapidly identify and mutate genes. We
further show that insertions can create alterations in gene expression,
and we have used this approach to investigate an amino acid oxidation
pathway in two important fungal phytopathogens.
Abnormal development of the hippocampal dentate gyrus in mice lacking the CXCR4 chemokine receptor
Authors: Lu Meiling , Grove Elizabeth A. , Miller Richard J.
Published: July 2002

We investigated the role of the CXCR4 chemokine receptor in development of the mouse hippocampus. CXCR4 mRNA is expressed at sites of neuronal and progenitor cell migration in the hippocampus at late embryonic and early postnatal ages. mRNA for stromal cell-derived factor 1 (SDF-1), the only known ligand for the CXCR4 receptor, is expressed close to these migration sites, in the meninges investing the hippocampal primordium and the primordium itself. In mice engineered to lack the CXCR4 receptor, the morphology of the hippocampal dentate gyrus (DG) is dramatically altered. Gene expression markers for DG granule neurons and bromodeoxyuridine labeling of dividing cells revealed an underlying defect in the stream of postmitotic cells and secondary dentate progenitor cells that migrate toward and form the DG. In the absence of CXCR4, the number of dividing cells in the migratory stream and in the DG itself is reduced, and neurons appear to differentiate prematurely before reaching their target. Our findings indicate a role for the SDF-1/CXCR4 chemokine signaling system in DG morphogenesis. Finally, the DG is unusual as a site of adult neurogenesis. We find that both
CXCR4
and
SDF-1
are expressed in the adult DG, suggesting an ongoing role in DG morphogenesis.
CpG methylation as a mechanism for the regulation of E2F activity
Authors: Campanero Miguel R. , Armstrong Monica I. , Flemington Erik K.
Published: July 2002

Regulation of gene expression in mammals through methylation of cytosine residues at CpG dinucleotides is involved in the development and progression of tumors. Because many genes that are involved in the control of cell proliferation are regulated by members of the E2F family of transcription factors and because some E2F DNA-binding sites are methylated
in vivo
, we have investigated whether CpG methylation can regulate E2F functions. We show here that methylation of E2F elements derived from the dihydrofolate reductase, E2F1, and cdc2 promoters prevents the binding of all E2F family members tested (E2F1 through E2F5). In contrast, methylation of the E2F elements derived from the c-myc and c-myb promoters minimally affects the binding of E2F2, E2F3, E2F4, and E2F5 but significantly inhibits the binding of E2F1. Consistent with these studies, E2F3, but not E2F1, activates transcription through methylated E2F sites derived from the c-myb and c-myc genes whereas both E2F1 and E2F3 fail to transactivate a reporter gene that is under the control of a methylated dihydrofolate reductase E2F site. Together, these data illustrate a means through which E2F activity can be controlled.
Corticotropin-releasing factor receptor 2 is a tonic suppressor of vascularization
Authors: Bale Tracy L. , Giordano Frank J. , Hickey Reed P. , Huang Yan , Nath Anjali K. , Peterson Kirk L. , Vale Wylie W. , Lee Kuo-Fen
Published: July 2002

Angiogenesis is regulated by means of a balance between activators and inhibitors. However, little is known regarding the regulation of the quiescent state of adult vessels. Corticotropin-releasing factor receptor 2 (CRFR2) is found in both endothelial and smooth muscle cells (SMCs) in the vasculature, where its function has remained elusive. We have investigated the role of CRFR2 as a determinant of tissue vascularization by comparing control and CRFR2-deficient mice with immunohistological and morphometric techniques. To define the mechanisms responsible for CRFR2 inhibition of angiogenesis, we have also examined
in vitro
the effect of ligand activation on cell proliferation, cell cycle protein phosphorylation, and capillary tube formation. Our results demonstrate that mice deficient for CRFR2 become hypervascularized postnatally. Activation of this receptor
in vitro
results in reduced vascular endothelial growth factor (VEGF) release from SMCs, an inhibition of SMC proliferation, and inhibition of capillary tube formation in collagen gels. Treatment of a subcutaneously injected gel matrix with a CRFR2 agonist inhibits growth factor-induced vascularization. Western blots show that cell cycle retinoblastoma protein, which is essential for cell cycle progression, is decreased by CRFR2 agonist treatment in SMCs. These results suggest that CRFR2 is a critical component of a pathway necessary for tonic inhibition of adult neovascularization. CRFR2 may be a potential target for therapeutic modulation of angiogenesis in cancer and ischemic cardiovascular disease.
Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size
Authors: Graham Margaret E. , O'Callaghan Dermott W. , McMahon Harvey T. , Burgoyne Robert D.
Published: July 2002

Accumulating evidence suggests that the kinetics of release from single secretory vesicles can be regulated and that quantal size can be modified during fast kiss-and-run fusion. Multiple pathways for vesicle retrieval have been identified involving clathrin and dynamin. It has been unclear whether dynamin could participate in a fast kiss-and-run process to reclose a transient fusion pore and thereby limit vesicle release. We have disrupted dynamin function in adrenal chromaffin cells by expression of the amphiphysin Src-homology domain 3 (SH3) or by application of guanosine 5′-[γ-thio]triphosphate (GTPγS), and have monitored single vesicle release events, evoked by digitonin and Ca
2+
, by using carbon-fiber amperometry. Under both conditions, there was an increase in mean quantal size accompanying an increase in the half-width of amperometric spikes and a slowing of the fall time. These data suggest the existence of a dynamin-dependent process that can terminate vesicle release under basal conditions. Protein kinase C activation changed release kinetics and decreased quantal size by shortening the release period. The effects of phorbol ester treatment were not prevented by expression of the amphiphysin SH3 domain or by GTPγS suggesting the existence of alternative dynamin-independent process underlying fast kiss-and-run exocytosis.
Potentiation of capsaicin receptor activity by metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hyperalgesia
Authors: Tominaga Makoto , Wada Makoto , Masu Masayuki
Published: July 2002

The capsaicin (vanilloid) receptor, VR1, is a sensory
neuron-specific ion channel that serves as a polymodal detector of
pain-producing chemical and physical stimuli. It has been proposed that
ATP, released from different cell types, initiates the sensation of
pain by acting predominantly on nociceptive ionotropic purinoceptors
located on sensory nerve terminals. In this study, we examined the
effects of extracellular ATP on VR1. In cells expressing VR1, ATP
increased the currents evoked by capsaicin or protons through
activation of metabotropic P2Y
1
receptors in a protein
kinase C-dependent pathway. The involvement of
G
q/11
-coupled metabotropic receptors in the potentiation of
VR1 response was confirmed in cells expressing both VR1 and M1
muscarinic acetylcholine receptors. In the presence of ATP, the
temperature threshold for VR1 activation was reduced from 42°C to
35°C, such that normally nonpainful thermal stimuli (i.e., normal
body temperature) were capable of activating VR1. This represents a
novel mechanism through which the large amounts of ATP released from
damaged cells in response to tissue trauma might trigger the sensation
of pain.
O
-linkage of
N
-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability
Authors: Yang Xiaoyong , Su Kaihong , Roos Mark D. , Chang Qing , Paterson Andrew J. , Kudlow Jeffrey E.
Published: July 2002

The posttranslational modification of eukaryotic intracellular
proteins by
O
-linked
N
-acetylglucosamine
(
O
-GlcNAc) monosaccharides is essential for cell
viability, yet its precise functional roles are largely unknown.
O
-GlcNAc transferase utilizes UDP-GlcNAc, the end
product of hexosamine biosynthesis, to catalyze this modification. The
availability of UDP-GlcNAc correlates with glycosylation levels of
intracellular proteins as well as with transcriptional levels of some
genes. Meanwhile, transcription factors and RNA polymerase II can be
modified by
O
-GlcNAc. A linkage between transcription
factor
O
-GlcNAcylation and transcriptional regulation
therefore has been postulated. Here, we show that
O
-GlcNAcylation of a chimeric transcriptional activator
containing the second activation domain of Sp1 decreases its
transcriptional activity both in an
in vitro
transcription system and in living cells, which is in concert with our
observation that
O
-GlcNAcylation of Sp1 activation
domain blocks its
in vitro
and
in vivo
interactions with other Sp1 molecules and TATA-binding
protein-associated factor II 110. Furthermore, overexpression of
O
-GlcNAc transferase specifically inhibits
transcriptional activation by native Sp1 in cells. Thus, our studies
provide direct evidence that
O
-GlcNAcylation of
transcription factors is involved in transcriptional regulation.
Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26
Authors: Marguet Didier , Baggio Laurie , Kobayashi Takashi , Bernard Anne-Marie , Pierres Michel , Nielsen Per F. , Ribel Ulla , Watanabe Takeshi , Drucker Daniel J. , Wagtmann Nicolai
Published: July 2002

A subset of prolyl oligopeptidases, including dipeptidyl-peptidase IV (DPP IV or CD26, EC3.4.14.5), specifically cleave off N-terminal dipeptides from substrates having proline or alanine in amino acid position 2. This enzyme activity has been implicated in the regulation of the biological activity of multiple hormones and chemokines, including the insulinotropic peptides glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Targeted inactivation of the CD26 gene yielded healthy mice that have normal blood glucose levels in the fasted state, but reduced glycemic excursion after a glucose challenge. Levels of glucose-stimulated circulating insulin and the intact insulinotropic form of GLP-1 are increased in CD26−/−mice. A pharmacological inhibitor of DPP IV enzymatic activity improved glucose tolerance in wild-type, but not in CD26−/−, mice. This inhibitor also improved glucose tolerance in GLP-1 receptor−/−mice, indicating that CD26 contributes to blood glucose regulation by controlling the activity of GLP-1 as well as additional substrates. These data reveal a critical role for CD26 in physiological glucose homeostasis, and establish it as a potential target for therapy in type II diabetes.
Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels
Authors: Carrasco Antonio J. , Dzeja Petras P. , Alekseev Alexey E. , Pucar Darko , Zingman Leonid V. , Abraham M. Roselle , Hodgson Denice , Bienengraeber Martin , Puceat Michel , Janssen Edwin , Wieringa Be , Terzic Andre
Published: July 2002

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K
+
(K
ATP
) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with K
ATP
channel-dependent membrane excitability remains elusive. Here, we identify that the response of K
ATP
channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the K
ATP
channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and K
ATP
channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with K
ATP
channels and associated functions.
Essential roles for four cytoplasmic intermediate filament proteins in
Caenorhabditis elegans
development
Authors: Karabinos Anton , Schmidt Henning , Harborth Jens , Schnabel Ralf , Weber Klaus
Published: July 2002

The structural proteins of the cytoplasmic intermediate filaments (IFs) arise in the nematode
Caenorhabditis elegans
from eight reported genes and an additional three genes now identified in the complete genome. With the use of double-stranded RNA interference (RNAi) for all 11
C. elegans
genes encoding cytoplasmic IF proteins, we observe phenotypes for the five genes
A1
,
A2
,
A3
,
B1
, and
C2
. These range from embryonic lethality (B1) and embryonic/larval lethality (A3) to larval lethality (A1 and A2) and a mild dumpy phenotype of adults (C2). Phenotypes A2 and A3 involve displaced body muscles and paralysis. They probably arise by reduction of hypodermal IFs that participate in the transmission of force from the muscle cells to the cuticle. The B1 phenotype has multiple morphogenetic defects, and the A1 phenotype is arrested at the L1 stage. Thus, at least four IF genes are essential for
C. elegans
development. Their RNAi phenotypes are lethal defects due to silencing of single IF genes. In contrast to
C. elegans
, no IF genes have been identified in the complete
Drosophila
genome, posing the question of how
Drosophila
can compensate for the lack of these proteins, which are essential in mammals and
C. elegans
. We speculate that the lack of IF proteins in
Drosophila
can be viewed as cytoskeletal alteration in which, for instance, stable microtubules, often arranged as bundles, substitute for cytoplasmic IFs.
Transposon stability and a role for conjugational transfer in adaptive mutability
Authors: Godoy V. G. , Fox M. S.
Published: July 2002

Lac
+
revertants of
Escherichia
coli
that occur after prolonged nonlethal selection display a high frequency of transposon loss when the transposon Tn
10
and the reverting
lacI33
allele are linked on an F′128 episome. As many as 20% of the Lac
+
revertants are sensitive to tetracycline, about half because of transposon loss, nearly all by precise excision, and the remainder because of amplification of both the transposon and the linked
lac
allele. Lethality of the amplified products in the presence of tetracycline is a peculiarity of the
tetA
gene at high gene dosage. The selective conditions on lactose medium result in 10% transposon-free revertants, whether or not a requirement for conjugal DNA transfer is imposed. In addition, a similar fraction, about 5% of Lac
−
unreverted colonies that are products of transfer between cells experiencing nonlethal selection are also tetracycline-sensitive, and all are attributable to loss of the Tn
10
transposon. These results suggest the possibility that the high frequency of transposon loss is a consequence of conjugal transfer, making this loss a marker for that transfer. We suggest that conjugal DNA transfer may be a prominent feature in the mutability process that occurs during nonlethal selection and that the subset of bacteria displaying hypermutability are those that experience such transfer.
Endothelial cell surface F
1
-F
O
ATP synthase is active in ATP synthesis and is inhibited by angiostatin
Authors: Moser Tammy L. , Kenan Daniel J. , Ashley Timothy A. , Roy Julie A. , Goodman Michael D. , Misra Uma K. , Cheek Dennis J. , Pizzo Salvatore V.
Published: July 2002

Angiostatin blocks tumor angiogenesis
in vivo
,
almost certainly through its demonstrated ability to block endothelial
cell migration and proliferation. Although the mechanism of angiostatin
action remains unknown, identification of F
1
-F
O
ATP synthase as the major angiostatin-binding site on the endothelial
cell surface suggests that ATP metabolism may play a role in the
angiostatin response. Previous studies noting the presence of
F
1
ATP synthase subunits on endothelial cells and certain
cancer cells did not determine whether this enzyme was functional in
ATP synthesis. We now demonstrate that all components of the
F
1
ATP synthase catalytic core are present on the
endothelial cell surface, where they colocalize into discrete punctate
structures. The surface-associated enzyme is active in ATP synthesis as
shown by dual-label TLC and bioluminescence assays. Both ATP synthase
and ATPase activities of the enzyme are inhibited by angiostatin as
well as by antibodies directed against the α- and β-subunits of ATP
synthase in cell-based and biochemical assays. Our data suggest that
angiostatin inhibits vascularization by suppression of
endothelial-surface ATP metabolism, which, in turn, may regulate
vascular physiology by established mechanisms. We now have shown that
antibodies directed against subunits of ATP synthase exhibit
endothelial cell-inhibitory activities comparable to that of
angiostatin, indicating that these antibodies function as angiostatin
mimetics.
Mitochondria as ATP consumers: Cellular treason in anoxia
Authors: St-Pierre Julie , Brand Martin D. , Boutilier Robert G.
Published: July 2002

In anoxia, mitochondria change from being ATP producers to potentially powerful ATP consumers. This change occurs, because the mitochondrial F1F0-ATPase begins to hydrolyze ATP to avoid the collapse of the proton motive force. Species that can survive prolonged periods of O2lack must limit such ATP use; otherwise, this process would dominate glycolytic metabolism and threaten ATP delivery to essential ATP-consuming processes of the cell (e.g., ion-motive ATPases). There are two ways to limit ATP hydrolysis by the F1F0-ATPase, namely (i) reduction of the proton conductance of the mitochondrial inner membrane and (ii) inhibition of the enzyme. We assessed these two possibilities by using intact mitochondria isolated from the skeletal muscle of anoxia-tolerant frogs. Our results show that proton conductance is unaltered between normoxia and anoxia. However, ATP use by the F1F0-ATPase is limited in anoxia by a profound inhibition of the enzyme. Even so, ATP use by the F1F0-ATPase might account for ≈9% of the ATP turnover in anoxic frog skeletal muscle.
Angiogenic role for glycodelin in tumorigenesis
Authors: Song Mingqing , Ramaswamy Sreemathy , Ramachandran Sumathi , Flowers Lisa C. , Horowitz Ira R. , Rock John A. , Parthasarathy Sampath
Published: July 2002

Angiogenesis plays an important role in neovascularization in tumors. Glycodelin, a hormone-responsive protein, has been detected in tumors of reproductive organs and is found in high levels in the plasma of subjects with gynecological malignancies. Glycodelin is also found in the endothelial cells of the umbilical cord and in the blood vessels of tumors. In this study, we tested whether glycodelin-rich amniotic fluid and a synthetic peptide derived from the sequence of glycodelin peptide (Gp) might promote angiogenic response by examining the migration and tube formation in human umbilical cord vein endothelial cells (HUVECs). Increased migration and tube formation of HUVECs were found in the presence of amniotic fluid and Gp, and this increase was blocked by antibody to Gp and by an anti-vascular endothelial growth factor (VEGF) antibody, suggesting that the angiogenic effects of glycodelin might be mediated by VEGF. The results also showed that Gp significantly increased the release of VEGF protein and mRNA expression in HUVECs, RL-95 (human endometrial carcinoma cells), OVCAR-3 (human ovarian adenocarcinoma cells), EM42 (human endometrial epithelial cells), THP-1 (human monocyte), and MCF-7 and MDA-MB-231 (human breast adenocarcinoma cells) cell lines. VEGF receptor Fit-1 mRNA expression in HUVECs was also increased in the presence of Gp. These findings, together with the suggestion from the literature that glycodelin may have immunosuppressive properties, suggest that glycodelin might play an important role in neovascularization during embryogenesis and tumor development.
Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB
Authors: Ganster Raymond W. , Taylor Bradley S. , Shao Lifang , Geller David A.
Published: July 2002

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between −5.2 and −6.1 kb in the 5′-flanking hiNOS promoter DNA, which contains multiple nuclear factor κβ (NF-κB) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signaltransducer andactivator oftranscription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-α, interleukin-1β, and IFN-γ (TNF-α, IL-1β, and IFN-γ) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at −5.8 kb in the hiNOS promoter identifies a bifunctional NF-κB/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at −5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-κB activates hiNOS promoter–reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-κB function in these cells. These results indicate that both Stat 1 and NF-κB are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.
Insulin-like growth factor 1 regulates developing brain glucose metabolism
Authors: Cheng Clara M. , Reinhardt Rickey R. , Lee Wei-Hua , Joncas George , Patel Sonya C. , Bondy Carolyn A.
Published: July 2002

The brain has enormous anabolic needs during early postnatal development. This study presents multiple lines of evidence showing that endogenous brain insulin-like growth factor 1 (Igf1) serves an essential, insulin-like role in promoting neuronal glucose utilization and growth during this period. Brain 2-deoxy-
d
- [1-
14
C]glucose uptake parallels Igf1 expression in wild-type mice and is profoundly reduced in
Igf1
−/− mice, particularly in those structures where Igf1 is normally most highly expressed. 2-Deoxy-
d
- [1-
14
C]glucose is significantly reduced in synaptosomes prepared from
Igf1
−/− brains, and the deficit is corrected by inclusion of Igf1 in the incubation medium. The serine/threonine kinase Akt/PKB is a major target of insulin-signaling in the regulation of glucose transport via the facilitative glucose transporter (GLUT4) and glycogen synthesis in peripheral tissues. Phosphorylation of Akt and GLUT4 expression are reduced in
Igf1
−/− neurons. Phosphorylation of glycogen synthase kinase 3β and glycogen accumulation also are reduced in
Igf1
−/− neurons. These data support the hypothesis that endogenous brain Igf1 serves an anabolic, insulin-like role in developing brain metabolism.
Chiral exciton coupling of merocyanine dyes within a well defined hydrogen-bonded assembly
Authors: Prins Leonard J. , Thalacker Christoph , Würthner Frank , Timmerman Peter , Reinhoudt David N.
Published: July 2002

Multichromophoric hydrogen-bonded assemblies 1
3
⋅(BAR)
6
are studied that bear a remarkably close resemblance to commelinin, a naturally occurring assembly responsible for an intense blue color of flowers. The incorporated chromophores exhibit a hypsochromic shift in the UV/visible (Vis) absorption maximum (Δ λ
max
= 14 nm) compared with the free chromophores. In addition, the chiroptical properties of incorporated chromophores can be rationally controlled by changing the supramolecular chirality of the assembly. These properties have been used to study the stability of this type of assembly with UV and CD spectroscopy at concentrations far below the NMR sensitivity threshold (10
−4
M). The determined C
50%
values of 2–3 μM in benzene show the extremely high stability of these
hydrogen-bonded
assemblies.
Near-precise interchromosomal recombination and functional DNA topoisomerase II cleavage sites at
MLL
and
AF-4
genomic breakpoints in treatment-related acute lymphoblastic leukemia with t(4;11) translocation
Authors: Lovett Brian D. , Lo Nigro Luca , Rappaport Eric F. , Blair Ian A. , Osheroff Neil , Zheng Naiyu , Megonigal Maureen D. , Williams Wendy Reed , Nowell Peter C. , Felix Carolyn A.
Published: July 2002

We analyzed the der(11) and der(4) genomic breakpoint junctions of
a t(4;11) in the leukemia of a patient previously administered
etoposide and dactinomycin by molecular and biochemical approaches to
gain insights about the translocation mechanism and the relevant drug
exposure. The genomic breakpoint junctions were amplified by PCR.
Cleavage of DNA substrates containing the normal homologues of the
MLL
and
AF-4
translocation breakpoints
was examined
in vitro
upon incubation with human DNA
topoisomerase IIα and etoposide, etoposide catechol, etoposide
quinone, or dactinomycin. The der(11) and der(4) genomic breakpoint
junctions both involved
MLL
intron 6 and
AF-4
intron 3. Recombination was precise at the sequence
level except for the overall gain of a single templated nucleotide. The
translocation breakpoints in
MLL
and
AF-4
were DNA topoisomerase II cleavage sites. Etoposide and its
metabolites, but not dactinomycin, enhanced cleavage at these sites.
Assuming that DNA topoisomerase II was the mediator of the breakage,
processing of the staggered nicks induced by DNA topoisomerase II,
including exonucleolytic deletion and template-directed polymerization,
would have been required before ligation of the ends to generate the
observed genomic breakpoint junctions. These data are inconsistent with
a translocation mechanism involving interchromosomal recombination by
simple exchange of DNA topoisomerase II subunits and DNA-strand
transfer; however, consistent with reciprocal DNA topoisomerase II
cleavage events in
MLL
and
AF-4
in which
both breaks became stable, the DNA ends were processed and underwent
ligation. Etoposide and/or its metabolites, but not dactinomycin,
likely were the relevant exposures in this patient.
Polar targeting of
Shigella
virulence factor IcsA in Enterobacteriacae and
Vibrio
Authors: Charles Macarthur , Pérez Marisa , Kobil Jessica H. , Goldberg Marcia B.
Published: July 2002

Asymmetric localization is key to the proper function of certain
prokaryotic proteins important to virulence, chemotaxis, cell division,
development, motility, and adhesion.
Shigella
IcsA is
localized to the old pole of the bacterium, where it mediates assembly
of an actin tail inside infected mammalian cells. IcsA (VirG) is
essential to
Shigella
intracellular motility and
virulence. We used translational fusions between portions of IcsA and
the green fluorescent protein (GFP) to determine the regions of IcsA
that are necessary and sufficient for its targeting to the bacterial
old pole. An IcsA-GFP fusion that lacks a signal peptide localized to
the old pole, indicating that signal peptide-mediated secretion is not
required for polar localization. Two regions within IcsA were required
for localization of an IcsA-GFP fusion to the old pole. Further
characterization of these regions indicated that amino acids 1–104 and
507–620 were each independently sufficient for polar localization.
Finally, when expressed in
Escherichia coli
,
Salmonella typhimurium
,
Yersinia
pseudotuberculosis
, and
Vibrio cholerae
, each of
the two targeting regions localized to the pole, indicating that the
mechanism of polar targeting used by IcsA is present generally among
Enterobacteriacae and
Vibrio
.
Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor
Authors: Rodríguez-Manzaneque Juan Carlos , Lane Timothy F. , Ortega María Asunción , Hynes Richard O. , Lawler Jack , Iruela-Arispe M. Luisa
Published: July 2002

Growth of tumors and metastasis are processes known to require neovascularization. To ascertain the participation of the endogenous angiogenic inhibitor thrombospondin-1 (TSP1) in tumor progression, we generated mammary tumor-prone mice that either lack, or specifically overexpress, TSP1 in the mammary gland. Tumor burden and vasculature were significantly increased in TSP1-deficient animals, and capillaries within the tumor appeared distended and sinusoidal. In contrast, TSP1 overexpressors showed delayed tumor growth or lacked frank tumor development (20% of animals); tumor capillaries showed reduced diameter and were less frequent. Interestingly, absence of TSP1 resulted in increased association of vascular endothelial growth factor (VEGF) with its receptor VEGFR2 and higher levels of active matrix metalloproteinase-9 (MMP9), a molecule previously shown to facilitate both angiogenesis and tumor invasion.
In vitro
, enzymatic activation of proMMP9 was suppressed by TSP1. Together these results argue for a protective role of endogenous inhibitors of angiogenesis in tumor growth and implicate TSP1 in the
in vivo
regulation of metalloproteinase-9 activation and VEGF signaling.
HIV envelope gp120 activates human arterial smooth muscle cells
Authors: Schecter Alison D. , Berman Adriane B. , Yi Lin , Mosoian Arevik , McManus Carrie M. , Berman Joan W. , Klotman Mary E. , Taubman Mark B.
Published: July 2002

There have been increasing reports of acute coronary thrombotic events in patients with HIV. Although these clinical events have been attributed primarily to dyslipidemia associated with protease inhibitor therapy, autopsy studies in children with HIV suggest the presence of an underlying arteriopathy. This study demonstrates that the HIV envelope protein, gp120, activates human arterial smooth muscle cells to express tissue factor, the initiator of the coagulation cascade. The induction of tissue factor by gp120 is mediated by two biologically relevant coreceptors for HIV infection, CXCR4 and CCR5, and is also dependent on the presence of functional CD4. Induction of tissue factor by gp120 requires activation of mitogen-activating protein kinases, activation of protein kinase C, and generation of reactive oxygen species, signaling pathways that have protean effects on smooth muscle cell physiology. The activation of smooth muscle cells by gp120 may play an important role in the vascular, thrombotic, and inflammatory responses to HIV infection.
Targeted adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival in murine sepsis
Authors: Oberholzer Caroline , Oberholzer Andreas , Bahjat Frances R. , Minter Rebecca M. , Tannahill Cynthia L. , Abouhamze Amer , LaFace Drake , Hutchins Beth , Clare-Salzler Michael J. , Moldawer Lyle L.
Published: July 2002

Sepsis remains a significant clinical conundrum, and recent clinical trials with anticytokine therapies have produced disappointing results. Animal studies have suggested that increased lymphocyte apoptosis may contribute to sepsis-induced mortality. We report here that inhibition of thymocyte apoptosis by targeted adenovirus-induced thymic expression of human IL-10 reduced blood bacteremia and prevented mortality in sepsis. In contrast, systemic administration of an adenovirus expressing IL-10 was without any protective effect. Improvements in survival were associated with increases in Bcl-2 expression and reductions in caspase-3 activity and thymocyte apoptosis. These studies demonstrate that thymic apoptosis plays a critical role in the pathogenesis of sepsis and identifies a gene therapy approach for its therapeutic intervention.
Photoperiodic information acquired and stored
in vivo
is retained
in vitro
by a circadian oscillator, the avian pineal gland
Authors: Brandstätter Roland , Kumar Vinod , Abraham Ute , Gwinner Eberhard
Published: July 2002

Endogenous circadian rhythms have been described in a
wide range of organisms from prokaryotes to man. Although basic
circadian mechanisms at the molecular level are genetically fixed,
certain properties of circadian rhythms at the organismic level can be
modified by environmental conditions and subsequently retained for some
time, even in organisms shielded from 24-hr environmental variations.
To investigate the capacity of animals to acquire and store
photoperiodic information, we examined activity and melatonin rhythms
in house sparrows during synchronization to two different photoperiods
and during subsequent prolonged darkness. Under constant environmental
conditions, intact animals continued to have long feeding activity
times when previously exposed to long days and short feeding activity
times when previously exposed to short days. Correspondingly,
significantly different durations of elevated melatonin in the plasma
directly reflected the differences in night length during
synchronization as well as during prolonged darkness. Additionally, we
found a significant difference in the amplitude of the nocturnal
melatonin signal, which also was conserved in prolonged darkness. To
investigate whether the photoperiodic experience of an intact animal
can be “memorized” by an isolated component of its circadian
pacemaking system, we have investigated
in vitro
melatonin release during continuous darkness from explanted pineal
glands of house sparrows after
in vivo
synchronization
to two distinct photoperiods. Differences in the durations of elevated
melatonin occurred during the first two cycles in culture and a
difference in melatonin amplitude was detectable during the first night
in culture. Our data indicate that photoperiodic patterns imposed on
sparrows during
in vivo
synchronization can be
maintained as an internal representation of time within the isolated
pineal gland. Hence, the pineal gland, as one of the most significant
components of the songbird circadian pacemaker, not only has the
capacity to autonomously produce circadian rhythms of melatonin release
but also is capable of storing biologically meaningful information
experienced during previous cycles.
Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4
Authors: Visintin Alberto , Mazzoni Alessandra , Spitzer Jessica A. , Segal David M.
Published: July 2002

Toll-like receptor 4 (TLR4), the principal signaling receptor for lipopolysaccharide (LPS) in mammals, requires the binding of MD-2 to its extracellular domain for maximal responsiveness. MD-2 contains a leader sequence but lacks a transmembrane domain, and we asked whether it is secreted into the medium as an active protein. As a source of secreted MD-2 (sMD-2), we used culture supernatants from cells stably transduced with epitope-tagged human MD-2. We show that sMD-2 exists as a heterogeneous collection of large disulfide-linked oligomers formed from stable dimeric subunits and that concentrations of sMD-2 as low as 50 pM enhance the responsiveness of TLR4 reporter cells to LPS. An MD-2-like activity is also released by monocyte-derived dendritic cells from normal donors. When coexpressed, TLR4 indiscriminately associates in the endoplasmic reticulum/cis Golgi with different-sized oligomers of MD-2, and excess MD-2 is secreted into the medium. We conclude that normal and transfected cells secrete a soluble form of MD-2 that binds with high affinity to TLR4 and that could play a role in regulating responses to LPS and other pathogen-derived substancesin vivo.
Chromosomal variation in neurons of the developing and adult mammalian nervous system
Authors: Rehen Stevens Kastrup , McConnell Michael J. , Kaushal Dhruv , Kingsbury Marcy A. , Yang Amy H. , Chun Jerold
Published: July 2002

A basic assumption about the normal nervous system is that its neurons possess identical genomes. Here we present direct evidence for genomic variability, manifested as chromosomal aneuploidy, among developing and mature neurons. Analysis of mouse embryonic cerebral cortical neuroblasts
in situ
detected lagging chromosomes during mitosis, suggesting the normal generation of aneuploidy in these somatic cells. Spectral karyotype analysis identified ≈33% of neuroblasts as aneuploid. Most cells lacked one chromosome, whereas others showed hyperploidy, monosomy, and/or trisomy. The prevalence of aneuploidy was reduced by culturing cortical explants in medium containing fibroblast growth factor 2. Interphase fluorescence
in situ
hybridization on embryonic cortical cells supported the rate of aneuploidy observed by spectral karyotyping and detected aneuploidy in adult neurons. Our results demonstrate that genomes of developing and adult neurons can be different at the level of whole chromosomes.
Reafferent copies of imitated actions in the right superior temporal cortex
Authors: Iacoboni Marco , Koski Lisa M. , Brass Marcel , Bekkering Harold , Woods Roger P. , Dubeau Marie-Charlotte , Mazziotta John C. , Rizzolatti Giacomo
Published: July 2002

Imitation is a complex phenomenon, the neural mechanisms of which are still largely unknown. When individuals imitate an action that already is present in their motor repertoire, a mechanism matching the observed action onto an internal motor representation of that action should suffice for the purpose. When one has to copy a new action, however, or to adjust an action present in one's motor repertoire to a different observed action, an additional mechanism is needed that allows the observer to compare the action made by another individual with the sensory consequences of the same action made by himself. Previous experiments have shown that a mechanism that directly matches observed actions on their motor counterparts exists in the premotor cortex of monkeys and humans. Here we report the results of functional magnetic resonance experiments, suggesting that in the superior temporal sulcus, a higher order visual region, there is a sector that becomes active both during hand action observation and during imitation even in the absence of direct vision of the imitator's hand. The motor-related activity is greater during imitation than during control motor tasks. This newly identified region has all the requisites for being the region at which the observed actions, and the reafferent motor-related copies of actions made by the imitator, interact.
A kinetically significant intermediate in the folding of barnase
Authors: Fersht Alan R.
Published: July 2002

A series of studies on the small protein barnase in the 1990s
established it as a paradigm for protein folding in which there is a
kinetically important intermediate. But, a recent study in PNAS claims
that there are no stable intermediates on the folding pathway. I
summarize the evidence that proves that the folding kinetics of barnase
is inconsistent with the absence of a folding intermediate. I
reinterpret the major evidence presented against the intermediate (an
inflection in the unfolding limb of a chevron plot) and show that the
inflection is precisely what is predicted from the energy diagram for a
three-state reaction with a kinetically significant on-pathway
intermediate. The inflection is indicative of a change of rate
determining step from the formation to breakdown of an intermediate on
unfolding. Other evidence presented against the intermediate is, in
fact, consistent with a kinetically important intermediate. I show how
the complexities in the kinetics provide a means for measuring
otherwise unobtainable rate constants and provide a strategy for
mapping the structure of the early transition state in folding. Rather
than refute multistate kinetics, the presence of the inflection in the
unfolding plot constitutes a novel type of evidence for on-pathway
folding intermediates.
Bcl-2 interrupts the ceramide-mediated pathway of cell death.
Authors: Zhang J , Alter N , Reed J C , Borner C , Obeid L M , Hannun Y A
Published: July 2002

Ceramide, a product of sphingomyelin turn-over, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In this study, we examine the relationship between the ceramide-mediated pathway of growth suppression and the bcl-2 protooncogene. In ALL-697 leukemia cells, the addition of the chemotherapeutic agent vincristine resulted in a time-dependent growth suppression characterized by marked apoptosis. The effects of vincristine on cell death were preceded by a prolonged and sustained accumulation of endogenous ceramide levels reaching -10.4 pmol ceramide/nmol phospholipids at 12 hr following the addition of vincristine--an increase of 220% over vehicle-treated cells. Overexpression of bcl-2 resulted in near total protection of cell death in response to vincristine. However, the ceramide response to vincristine was not modulated by overexpression of bcl-2, indicating that bcl-2 does not interfere with ceramide formation. Overexpression of bcl-2 prevented apoptosis in response to ceramide, suggesting that bcl-2 acts at a point downstream of ceramide. On the other hand, bcl-2 did not interfere with the ability of ceramide to activate the retinoblastoma gene product or to induce cell cycle arrest, suggesting that the effects of ceramide on cell cycle arrest can be dissociated from the effects on apoptosis. These studies suggest that ceramide and bcl-2 partake in a common pathway of cell regulation. The results also cast ceramide as a gauge of cell injury rather than an "executor" of cell death with clearly dissociable biological outcomes of its action depending on downstream factors.
OB protein binds specifically to the choroid plexus of mice and rats.
Authors: Devos R , Richards J G , Campfield L A , Tartaglia L A , Guisez Y , van der Heyden J , Travernier J , Plaetinck G , Burn P
Published: July 2002

Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor.
Promotion of sleep mediated by the A2a-adenosine receptor and possible involvement of this receptor in the sleep induced by prostaglandin D2 in rats.
Authors: Satoh S , Matsumura H , Suzuki F , Hayaishi O
Published: July 2002

A 6-hr continuous infusion of 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenos ine (CGS21680), a selective A2a-adenosine agonist, into the subarachnoid space underlying the ventral surface region of the rostral basal forebrain, which has been defined as the prostaglandin (PG) D2-sensitive sleep-promoting zone, at rates of 0.02, 0.2, 2.0, and 12 pmol/min increased slow-wave sleep (SWS) and paradoxical sleep (PS) in a dose-dependent manner up to 183% and 202% of their respective baseline levels. The increments produced by the infusion of CGS21680 at 0.2 and 2.0 pmol/min were totally diminished when the rats had been pretreated with an i.p. injection of (E)-1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF17837; 30 mg/kg of body weight), a selective A2-adenosine antagonist. In contrast, the infusion of N6-cyclohexyladenosine (CHA), a selective A1-adenosine agonist, at 2 pmol/min significantly suppressed SWS before causing an increase in SWS, and a decrease in PS was also markedly visible. Essentially the same effects of CGS21680 and CHA were observed when these compounds were administered to the parenchymal region of the rostral basal forebrain through chronically implanted microdialysis probes. Thus, we clearly showed that stimulation of A2a-adenosine receptors in the rostral basal forebrain promotes SWS and PS. Furthermore, i.p. injections of KF17837 at 30 and 100 mg/kg of body weight dose-dependently attenuated the magnitude of the SWS increase produced by the infusion of PGD2 into the subarachnoid space of the sleep-promoting zone, thus indicating that the A2a-adenosine receptors are crucial in the sleep-promoting process triggered by PGD2.
Taste receptor-like cells in the rat gut identified by expression of alpha-gustducin.
Authors: Höfer D , Püschel B , Drenckhahn D
Published: July 2002

The alpha-subunit of the trimeric G-protein complex specific for taste receptor cells of the tongue, alpha-gustducin, is described here to be also expressed in the stomach and intestine. The alpha-gustducin-containing cells were identified as brush cells that are scattered throughout the surface epithelium of the gut and share structural features of taste receptor cells of the tongue. These findings provide clues to the long-sought molecular and cellular basis for chemoreception in the gut.