bluuebunny/crossref_metadata_2025 huggingface dataset:
The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase
Authors: Schneider Katja , Hövel Klaus , Witzel Kilian , Hamberger Björn , Schomburg Dietmar , Kombrink Erich , Stuible Hans-Peter
Published: July 2003

To reveal the structural principles determining substrate specificity of
4-coumarate:CoA ligase (4CL), the crystal structure of the phenylalanine
activation domain of gramicidin S synthetase was used as a template for
homology modeling. According to our model, 12 amino acid residues lining the
Arabidopsis
4CL isoform 2 (At4CL2) substrate binding pocket (SBP)
function as a signature motif generally determining 4CL substrate specificity.
We used this substrate specificity code to create At4CL2 gain-of-function
mutants. By increasing the space within the SBP we generated ferulic- and
sinapic acid-activating At4CL2 variants. Increasing the hydrophobicity of the
SBP resulted in At4CL2 variants with strongly enhanced conversion of cinnamic
acid. These enzyme variants are suitable tools for investigating and
influencing metabolic channeling mediated by 4CL. Knowledge of the 4CL
specificity code will facilitate the prediction of substrate preference of
numerous, still uncharacterized 4CL-like proteins.
Neural correlates of interindividual differences in the subjective experience of pain
Authors: Coghill Robert C. , McHaffie John G. , Yen Yi-Fen
Published: July 2003

Some individuals claim that they are very sensitive to pain, whereas others say that they tolerate pain well. Yet, it is difficult to determine whether such subjective reports reflect true interindividual experiential differences. Using psychophysical ratings to define pain sensitivity and functional magnetic resonance imaging to assess brain activity, we found that highly sensitive individuals exhibited more frequent and more robust pain-induced activation of the primary somatosensory cortex, anterior cingulate cortex, and prefrontal cortex than did insensitive individuals. By identifying objective neural correlates of subjective differences, these findings validate the utility of introspection and subjective reporting as a means of communicating a first-person experience.
A SNARE complex containing SGR3/AtVAM3 and ZIG/VTI11 in gravity-sensing cells is important for
Arabidopsis
shoot gravitropism
Authors: Yano Daisuke , Sato Masakazu , Saito Chieko , Sato Masa H. , Morita Miyo Terao , Tasaka Masao
Published: July 2003

Plants can sense the direction of gravity and change the growth orientation
of their organs. The molecular mechanisms of gravity sensing and signal
transduction during gravitropism are not well known. We have isolated several
shoot gravitropism
(
sgr
) mutants of
Arabidopsis
.
The
sgr3-1
mutant exhibits a reduced gravitropic response in the
inflorescence stems. In the inflorescence stems of
Arabidopsis
,
gravity is sensed in endodermal cells that contain sedimentable amyloplasts.
In
sgr3-1
, some amyloplasts in the endodermis failed to sediment in
the direction of gravity.
SGR3
encodes a syntaxin, AtVAM3, which had
previously been cloned as a homologue of yeast Vam3p. AtVAM3 is localized to
the prevacuolar compartment and vacuole and is suggested to function in
vesicle transport to the vacuole. We have also cloned another soluble
N
-ethylmaleimide-sensitive factor attachment protein receptor
(SNARE), ZIG/AtVTI11, a mutation that causes abnormal gravitropism. This
mutant displayed an abnormal distribution of amyloplasts in the endodermal
cells similar to that in
sgr3-1
. Endodermis-specific expression of
SGR3
and
ZIG
by using the
SCR
promoter could
complement the abnormal shoot gravitropism of each mutant.
Protein–protein interaction between AtVAM3 and AtVTI11 in the endodermal
cells was detected immunologically. The
sgr3-1
mutation appeared to
reduce the affinity of AtVAM3 for AtVTI11 or SYP5. These results suggest that
vesicle transport to the prevacuolar compartment/vacuole in the endodermal
cells, mediated by a specific SNARE complex containing AtVAM3 and AtVTI11,
plays an important role in shoot gravitropism.
Human topoisomerase I promotes HIV-1 proviral DNA synthesis: Implications for the species specificity and cellular tropism of HIV-1 infection
Authors: Shoya Yuko , Tokunaga Kenzo , Sawa Hirofumi , Maeda Masae , Ueno Tomonori , Yoshikawa Tomoki , Hasegawa Hideki , Sata Tetsutaro , Kurata Takeshi , Hall William W. , Cullen Bryan R. , Takahashi Hidehiro
Published: July 2003

Although HIV type 1 (HIV-1) cannot efficiently replicate in simian cells, the mechanism(s) involved in the restriction of virus tropism remain unclear. To investigate this, we have focused on the identification of human cellular factors that can influence the infectivity of HIV-1 derived from African green monkey producer cells. Whereas the infectivity of HIV-1 derived from such cells was only 10–15% of that of human cell-derived virus, expression of human topoisomerase I in the African green monkey cells resulted in a 5-fold increase of the infectivity of progeny HIV-1 virions. Replacement of glutamate-236 and asparagine-237 of human topoisomerase I with the corresponding residues (aspartate and serine, respectively) of the African green monkey enzyme abolished this enhancement of HIV-1 infectivity. This positive effect of human topoisomerase I expression in the African green monkey producer cells seemed to result from the promotion of HIV-1 cDNA synthesis. Thus, human topoisomerase I plays an important role in HIV-1 replication and infectivity, and differences in the species specificity of HIV-1 infection can at least in part be attributed to differences in topoisomerase I activities.
SLC5A8
, a sodium transporter, is a tumor suppressor gene silenced by methylation in human colon aberrant crypt foci and cancers
Authors: Li Hui , Myeroff Lois , Smiraglia Dominic , Romero Michael F. , Pretlow Theresa P. , Kasturi Lakshmi , Lutterbaugh James , Rerko Ronald M. , Casey Graham , Issa Jean-Pierre , Willis Joseph , Willson James K. V. , Plass Christoph , Markowitz Sanford D.
Published: July 2003

We identify a gene,
SLC5A8
, and show it is a candidate tumor
suppressor gene whose silencing by aberrant methylation is a common and early
event in human colon neoplasia. Aberrant DNA methylation has been implicated
as a component of an epigenetic mechanism that silences genes in human
cancers. Using restriction landmark genome scanning, we performed a global
search to identify genes that would be aberrantly methylated at high frequency
in human colon cancer. From among 1,231 genomic
Not
I sites assayed,
site 3D41 was identified as methylated in 11 of 12 colon cancers profiled.
Site 3D41 mapped to exon 1 of
SLC5A8
, a transcript that we assembled.
In normal colon mucosa we found that
SLC5A8
exon 1 is unmethylated
and
SLC5A8
transcript is expressed. In contrast,
SLC5A8
exon
1 proved to be aberrantly methylated in 59% of primary colon cancers and 52%
of colon cancer cell lines.
SLC5A8
exon 1 methylated cells were
uniformly silenced for SLC5A8 expression, but reactivated expression on
treatment with a demethylating drug, 5-azacytidine. Transfection of
SLC5A8
suppressed colony growth in each of three SLC5A8-deficient
cell lines, but showed no suppressive effect in any of three SLC5A8-proficient
cell lines.
SLC5A8
exon 1 methylation is an early event, detectable
in colon adenomas, and in even earlier microscopic colonic aberrant crypt
foci. Structural homology and functional testing demonstrated that SLC5A8 is a
member of the family of sodium solute symporters, which are now added as a
class of candidate colon cancer suppressor genes.
A
Pseudomonas syringae
type III effector suppresses cell wall-based extracellular defense in susceptible
Arabidopsis
plants
Authors: Hauck Paula , Thilmony Roger , He Sheng Yang
Published: July 2003

Bacterial effector proteins secreted through the type III secretion system
(TTSS) play a crucial role in causing plant and human diseases. Although the
ability of type III effectors to trigger defense responses in resistant plants
is well understood, the disease-promoting functions of type III effectors in
susceptible plants are largely enigmatic. Previous microscopic studies suggest
that in susceptible plants the TTSS of plant-pathogenic bacteria transports
suppressors of a cell wall-based plant defense activated by the TTSS-defective
hrp
mutant bacteria. However, the identity of such suppressors has
remained elusive. We discovered that the
Pseudomonas syringae
TTSS
down-regulated the expression of a set of
Arabidopsis
genes encoding
putatively secreted cell wall and defense proteins in a salicylic
acid-independent manner. Transgenic expression of AvrPto repressed a similar
set of host genes, compromised defense-related callose deposition in the host
cell wall, and permitted substantial multiplication of an
hrp
mutant.
AvrPto is therefore one of the long postulated suppressors of an salicylic
acid-independent, cell wall-based defense that is aimed at
hrp
mutant
bacteria.
The first tunicate from the Early Cambrian of South China
Authors: Chen Jun-Yuan , Huang Di-Ying , Peng Qing-Qing , Chi Hui-Mei , Wang Xiu-Qiang , Feng Man
Published: July 2003

Here we report the discovery of eight specimens of an Early Cambrian fossil
tunicate
Shankouclava
near Kunming (South China). The tunicate
identity of this organism is supported by the presence of a large and
perforated branchial basket, a sac-like peri-pharyngeal atrium, an oral siphon
with apparent oral tentacles at the basal end of the siphonal chamber, perhaps
a dorsal atrial pore, and an elongated endostyle on the mid-ventral floor of
the pharynx. As in most modern tunicates, the gut is simple and U-shaped, and
is connected with posterior end of the pharynx at one end and with an atrial
siphon at the other, anal end.
Shankouclava
differs from
Cheungkongella
, which was previously called a tunicate. Based on new,
more complete “
Cheungkongella
” specimens that show
branching tentacles, this form may be a lophophorate, and in any case is not a
tunicate.
A major role for carbon monoxide as an endogenous hyperpolarizing factor in the gastrointestinal tract
Authors: Farrugia Gianrico , Lei Sha , Lin Xue , Miller Steven M. , Nath Karl A. , Ferris Christopher D. , Levitt Michael , Szurszewski Joseph H.
Published: July 2003

Carbon monoxide (CO) is proposed as a physiological messenger. CO activates
cGMP and has a direct effect on potassium channels. Both actions of CO lead to
hyperpolarization of a cell's resting membrane potential, suggesting that CO
may function as a hyperpolarizing factor, although direct evidence is still
lacking. Here we take advantage of the known membrane potential gradient that
exists in the muscle layers of the gastrointestinal tract to determine whether
CO is an endogenous hyperpolarizing factor. We find that heme oxygenase-2-null
mice have depolarized smooth muscle cells and that the membrane potential
gradient in the gut is abolished. Exogenous CO hyperpolarizes the membrane
potential. Regions of the canine gastrointestinal tract that are more
hyperpolarized generate more CO and have higher heme oxygenase activity than
more depolarized regions. Our results suggest that CO is a critical
hyperpolarizing factor required for the maintenance of intestinal smooth
muscle membrane potential and gradient.
Complete genome sequence of the marine planctomycete
Pirellula
sp. strain 1
Authors: Glöckner F. O. , Kube M. , Bauer M. , Teeling H. , Lombardot T. , Ludwig W. , Gade D. , Beck A. , Borzym K. , Heitmann K. , Rabus R. , Schlesner H. , Amann R. , Reinhardt R.
Published: July 2003

Pirellula
sp. strain 1 (“
Rhodopirellula
baltica
”) is a marine representative of the globally distributed
and environmentally important bacterial order
Planctomycetales
. Here
we report the complete genome sequence of a member of this independent phylum.
With 7.145 megabases,
Pirellula
sp. strain 1 has the largest circular
bacterial genome sequenced so far. The presence of all genes required for
heterolactic acid fermentation, key genes for the interconversion of C1
compounds, and 110 sulfatases were unexpected for this aerobic heterotrophic
isolate. Although
Pirellula
sp. strain 1 has a proteinaceous cell
wall, remnants of genes for peptidoglycan synthesis were found. Genes for
lipid A biosynthesis and homologues to the flagellar L- and P-ring protein
indicate a former Gram-negative type of cell wall. Phylogenetic analysis of
all relevant markers clearly affiliates the
Planctomycetales
to the
domain
Bacteria
as a distinct phylum, but a deepest branching is not
supported by our analyses.
Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase
Authors: Kuwabara Yoshimitsu , Nishino Tomoko , Okamoto Ken , Matsumura Tomohiro , Eger Bryan T. , Pai Emil F. , Nishino Takeshi
Published: July 2003

In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH)
but can be readily converted to its oxidase form (XO) either by proteolysis or
modification of cysteine residues. The crystal structures of bovine milk XDH
and XO demonstrated that atoms in the highly charged active-site loop
(Gln-423–Lys-433) around the FAD cofactor underwent large dislocations
during the conversion, blocking the approach of the NAD
+
substrate
to FAD in the XO form as well as changing the electrostatic environment around
FAD. Here we identify a unique cluster of amino acids that plays a dual role
by forming the core of a relay system for the XDH/XO transition and by gating
a solvent channel leading toward the FAD ring. A more detailed structural
comparison and site-directed mutagenesis analysis experiments showed that
Phe-549, Arg-335, Trp-336, and Arg-427 sit at the center of a relay system
that transmits modifications of the linker peptide by cysteine oxidation or
proteolytic cleavage to the active-site loop (Gln-423–Lys-433). The
tight interactions of these residues are crucial in the stabilization of the
XDH conformation and for keeping the solvent channel closed. Both oxidative
and proteolytic generation of XO effectively leads to the removal of Phe-549
from the cluster causing a reorientation of the bulky side chain of Trp-336,
which then in turn forces a dislocation of Arg-427, an amino acid located in
the active-site loop. The conformational change also opens the gate for the
solvent channel, making it easier for oxygen to reach the reduced FAD in
XO.
Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1 receptor
Authors: Girnita Leonard , Girnita Ada , Larsson Olle
Published: July 2003

Recently, p53 was demonstrated to affect the expression of the insulin-like
growth factor 1 receptor (IGF-1R), a receptor tyrosine kinase that plays a
crucial role in growth and survival of cancer cells. However, the underlying
mechanisms for interaction between p53 and IGF-1R are still not fully
understood. One of the challenging questions remaining to be answered is why
the wild-type p53, which
per se
represses the transcription of the
IGF-1R
gene, in overexpressed form is necessary for a high IGF-1R
expression. In this study, we show that inhibition of p53 causes
ubiquitination and down-regulation, through increased degradation, of the
IGF-1R in human malignant melanoma cells. This effect, which was independent
of the p53 status (i.e., wild type or mutated), was prevented if Mdm2 was
coinhibited. Similar results were obtained in UV-irradiated human melanocytes
(harboring wild-type p53), in which level of the IGF-1R increased after
up-regulation of p53. Interestingly, the basal ubiquitination of the IGF-1R in
untreated cells also depended on Mdm2. We could prove that Mdm2 physically
associates with IGF-1R and that Mdm2 causes IGF-1R ubiquitination in an
in
vitro
assay. Taken together our data provide evidence that Mdm2 serves as
a ligase in ubiquitination of the IGF-1R and thereby causes its degradation by
the proteasome system. Consequently, by sequestering Mdm2 in the cell nuclei,
the level of p53 may indirectly influence the expression of IGF-1R. This role
of Mdm2 and p53 represents an unexpected mechanism for the regulation of
IGF-1R and cell growth.
Birds defend trees from herbivores in a Neotropical forest canopy
Authors: Van Bael Sunshine A. , Brawn Jeffrey D. , Robinson Scott K.
Published: July 2003

Most forest birds include arthropods in their diet, sometimes specializing
on arthropods that consume plant foliage. Experimental tests of whether bird
predation on arthropods can reduce plant damage, however, are few and
restricted to relatively low-diversity systems. Here, we describe an
experimental test in a diverse tropical forest of whether birds indirectly
defend foliage from arthropod herbivores. We also compare how the indirect
effects of bird predation vary with different levels of foliage productivity
in the canopy vs. the understory. For three Neotropical tree species, we
observed that birds decreased local arthropod densities on canopy branches and
reduced consequent damage to leaves. In contrast, we observed no evidence of
bird–arthropod limitation on conspecific saplings in the less productive
understory of the same forest. Our results support theory that predicts
trophic cascades where productivity is high and suggest that birds play an
important role in Neotropical communities by means of their indirect defense
of some canopy tree species.
The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeastdna2mutants
Authors: Imamura Osamu , Campbell Judith L.
Published: July 2003

Bloom syndrome is a disorder of profound and early cancer predisposition in which cells become hypermutable, exhibit high frequency of sister chromatid exchanges, and show increased micronuclei.BLM, the gene mutated in Bloom syndrome, has been cloned previously, and the BLM protein is a member of the RecQ family of DNA helicases. Many lines of evidence suggest thatBLMis involved either directly in DNA replication or in surveillance during DNA replication, but its specific roles remain unknown. Here we show thathBLMcan suppress both the temperature-sensitive growth defect and the DNA damage sensitivity of the yeast DNA replication mutantdna2-1. Thedna2-1mutant is defective in a helicase-nuclease that is required either to coordinate with the crucialSaccharomyces cerevisiae(sc) FEN1 nuclease in Okazaki fragment maturation or to compensate for scFEN1 when its activity is impaired. We show that human BLM interacts with both scDna2 and scFEN1 by using coimmunoprecipitation from yeast extracts, suggesting that human BLM participates in the same steps of DNA replication or repair as scFEN1 and scDna2.
Understanding the function–structure and function–mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis
Authors: Kato Shunsuke , Han Shuang-Yin , Liu Wen , Otsuka Kazunori , Shibata Hiroyuki , Kanamaru Ryunosuke , Ishioka Chikashi
Published: July 2003

Inactivation of the tumor suppressor p53 by missense mutations is the most
frequent genetic alteration in human cancers. The common missense mutations in
the
TP53
gene disrupt the ability of p53 to bind to DNA and
consequently to transactivate downstream genes. However, it is still not fully
understood how a large number of the remaining mutations affect p53 structure
and function. Here, we used a comprehensive site-directed mutagenesis
technique and a yeast-based functional assay to construct, express, and
evaluate 2,314 p53 mutants representing all possible amino acid substitutions
caused by a point mutation throughout the protein (5.9 substitutions per
residue), and correlated p53 function with structure- and tumor-derived
mutations. This high-resolution mutation analysis allows evaluation of
previous predictions and hypotheses through interrelation of function,
structure and mutation.
Use of in
vivo
-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with
Vibrio cholerae
Authors: Hang Long , John Manohar , Asaduzzaman Muhammad , Bridges Emily Anna , Vanderspurt Cecily , Kirn Thomas J. , Taylor Ronald K. , Hillman Jeffrey D. , Progulske-Fox Ann , Handfield Martin , Ryan Edward T. , Calderwood Stephen B.
Published: July 2003

In vivo
-induced antigen technology is a method to identify
proteins expressed by pathogenic bacteria during human infection. Sera from 10
patients convalescing from cholera infection in Bangladesh were pooled,
adsorbed against
in vitro
-grown El Tor
Vibrio cholerae
O1,
and used to probe a genomic expression library in
Escherichia coli
constructed from El Tor
V. cholerae
O1 strain N16961. We identified
38 positive clones in the screen, encoding pili (PilA and TcpA), cell membrane
proteins (PilQ, MshO, MshP, and CapK), methyl-accepting chemotaxis proteins,
chemotaxis and motility proteins (CheA and CheR), a quorum-sensing protein
(LuxP), and four hypothetical proteins. Analysis of immune responses to
purified PilA and TcpA in individual patients demonstrated that the majority
seroconverted to these proteins, confirming results with pooled sera. These
results suggest that PilA and its outer membrane secretin, PilQ, are expressed
during human infection and may be involved in colonization of the
gastrointestinal tract. These results also demonstrate substantial immune
responses to TcpA in patients infected with El Tor
V. cholerae
O1.
In vivo
-induced antigen technology provides a simple method for
identifying microbial proteins expressed during human infection, but not
during
in vitro
growth.
Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity
Authors: Rougeot Catherine , Messaoudi Michaël , Hermitte Véronique , Rigault Anne Gaëlle , Blisnick Thierry , Dugave Christophe , Desor Didier , Rougeon François
Published: July 2003

Sialorphin is an exocrine and endocrine signaling mediator, which has been
identified by a genomic approach. It is synthesized predominantly in the
submandibular gland and prostate of adult rats in response to androgen
steroids and is released locally and systemically in response to stress. We
now demonstrate that the cell surface molecule to which sialorphin binds
in vivo
in the rat kidney is the membrane-anchored neutral
endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in
nervous and peripheral tissues, as it turns off several peptide-signaling
events at the cell surface. We show that sialorphin prevents spinal and renal
NEP from breaking down its two physiologically relevant substrates, substance
P and Met-enkephalin
in vitro
. Sialorphin inhibited the breakdown of
substance P with an IC
50
of 0.4–1 μM and behaved as a
competitive inhibitor.
In vivo
, i.v. sialorphin elicited potent
antinociceptive responses in two behavioral rat models of injury-induced acute
and tonic pain, the pin-pain test and formalin test. The analgesia induced by
100–200 μg/kg doses of sialorphin required the activation of μ-
and δ-opioid receptors, consistent with the involvement of endogenous
opioid receptors in enkephalinergic transmission. We conclude that sialorphin
protects endogenous enkephalins released after nociceptive stimuli by
inhibiting NEP
in vivo
. Sialorphin is a natural systemically active
regulator of NEP activity. Furthermore, our study provides evidence that it is
a physiological modulator of pain perception after injury and might be the
progenitor of a new class of therapeutic molecules.
RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation
Authors: Mazan-Mamczarz Krystyna , Galbán Stefanie , de Silanes Isabel López , Martindale Jennifer L. , Atasoy Ulus , Keene Jack D. , Gorospe Myriam
Published: July 2003

Exposure to short-wavelength UV light (UVC) strongly induces p53
expression. In human RKO colorectal carcinoma cells, this increase was not due
to elevated p53 mRNA abundance, cytoplasmic export of p53 mRNA, or
UVC-triggered stabilization of the p53 protein. Instead, p53 translation was
potently enhanced after UVC irradiation. The 3′ UTR of p53 was found to
be a target of the RNA-binding protein HuR in a UVC-dependent manner
in
vitro
and
in vivo
. HuR-overexpressing RKO cells displayed
elevated p53 levels, whereas cells expressing reduced HuR showed markedly
diminished p53 abundance and p53 translation. Our results demonstrate a role
for HuR in binding to the p53 mRNA and enhancing its translation.
Functional reconstitution of COPI coat assembly and disassembly using chemically defined components
Authors: Reinhard Constanze , Schweikert Michael , Wieland Felix T. , Nickel Walter
Published: July 2003

Coat protein I (COPI)-coated transport vesicles mediate protein and lipid
transport in the early secretory pathway. The basic machinery required for the
formation of these transport intermediates has been elucidated based on the
reconstitution of COPI-coated vesicle formation from chemically defined
liposomes. In this experimental system, the coat components coatomer and
GTP-bound ADP-ribosylation factor (ARF), as well as p23 as a membrane-bound
receptor for COPI coat proteins, were shown to be both necessary and
sufficient to promote COPI-coated vesicle formation. Based on biochemical and
ultrastructural analyses, we now demonstrate that the catalytic domain of
ARF-GTPase-activating protein (GAP) alone is sufficient to initiate uncoating
of liposome-derived COPI-coated vesicles. By contrast, ARF-GAP activity is not
required for COPI coat assembly and, therefore, does not seem to represent an
essential coat component of COPI vesicles as suggested recently [Yang, J. S.,
Lee, S. Y., Gao, M., Bourgoin, S., Randazzo, P. A.,
et al.
(2002)
J. Cell Biol.
159, 69–78]. Thus, a complete round of COPI coat
assembly and disassembly has been reconstituted with purified components
defining the core machinery of COPI vesicle biogenesis.
R5 HIV productively infects Langerhans cells, and infection levels are regulated by compoundCCR5polymorphisms
Authors: Kawamura Tatsuyoshi , Gulden Forrest O. , Sugaya Makoto , McNamara David T. , Borris Debra L. , Lederman Michael M. , Orenstein Jan M. , Zimmerman Peter A. , Blauvelt Andrew
Published: July 2003

Langerhans cells (LCs) are suspected to be initial targets for HIV after sexual exposure (by becoming infected or by capturing virus). Here, productive R5 HIV infection of LCex vivoand LC-mediated transmission of virus to CD4+T cells were both found to depend on CCR5. By contrast, infection of monocyte-derived dendritic cells and transfer of infection from monocyte-derived dendritic cells to CD4+T cells were mediated by CCR5-dependent as well as DC-specific ICAM-3-grabbing nonintegrin-dependent pathways. Furthermore, in 62 healthy individuals, R5 HIV infection levels in LCsex vivowere associated withCCR5genotype. Specifically, genotyping forORFΔ32revealed that LCs isolated fromORFΔ32/wtindividuals were significantly less susceptible to HIV when compared with LCs isolated fromORFwt/wtindividuals (P= 0.016). Strikingly, further genetic analyses of theA-2459G CCR5promoter polymorphism inORFΔ32/wtheterozygous individuals revealed that LCs isolated from -2459A/G+ORFΔ32/wtindividuals were markedly less susceptible to HIV than were LCs from -2459A/A+ORFΔ32/wtindividuals (P= 0.012). Interestingly, these genetic susceptibility data in LCs parallel those of genetic susceptibility studies performed in cohorts of HIV-infected individuals. Thus, we suggest that CCR5-mediated infection of LCs, and not capture of virus by LCs, provides a biologic basis for understanding certain aspects of host genetic susceptibility to initial HIV infection.
Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway
Authors: Lehnardt Seija , Massillon Leon , Follett Pamela , Jensen Frances E. , Ratan Rajiv , Rosenberg Paul A. , Volpe Joseph J. , Vartanian Timothy
Published: July 2003

Innate immunity is an evolutionarily ancient system that provides organisms with immediately available defense mechanisms through recognition of pathogen-associated molecular patterns. We show that in the CNS, specific activation of innate immunity through a Toll-like receptor 4 (TLR4)-dependent pathway leads to neurodegeneration. We identify microglia as the major lipopolysaccharide (LPS)-responsive cell in the CNS. TLR4 activation leads to extensive neuronal deathin vitrothat depends on the presence of microglia. LPS leads to dramatic neuronal loss in cultures prepared from wild-type mice but does not induce neuronal injury in CNS cultures derived fromtlr4mutant mice. In anin vivomodel of neurodegeneration, stimulating the innate immune response with LPS converts a subthreshold hypoxic-ischemic insult from no discernable neuronal injury to severe axonal and neuronal loss. In contrast, animals bearing a loss-of-function mutation in thetlr4gene are resistant to neuronal injury in the same model. The present study demonstrates a mechanistic link among innate immunity, TLRs, and neurodegeneration.
Mutations in the clathrin-assembly gene
Picalm
are responsible for the hematopoietic and iron metabolism abnormalities in
fit1
mice
Authors: Klebig Mitchell L. , Wall Melissa D. , Potter Mark D. , Rowe Erica L. , Carpenter Donald A. , Rinchik Eugene M.
Published: July 2003

Recessive
N
-ethyl-
N
-nitrosourea (ENU)-induced mutations
recovered at the fitness-1 (
fit1
) locus in mouse chromosome 7 cause
hematopoietic abnormalities, growth retardation, and shortened life span, with
varying severity of the defects in different alleles. Abnormal iron
distribution and metabolism and frequent scoliosis have also been associated
with an allele of intermediate severity (

fit1
4R

). We
report that

fit1
4R

, as well as the most severe

fit1
5R

allele, are nonsense point mutations in the mouse
ortholog of the human phosphatidylinositol-binding clathrin assembly protein
(
PICALM
) gene, whose product is involved in clathrin-mediated
endocytosis. A variety of leukemias and lymphomas have been associated with
translocations that fuse human
PICALM
with the putative transcription
factor gene AF10. The

Picalm
fit1–5R

and

Picalm
fit1–4R

mutations are splice-donor alterations
resulting in transcripts that are less abundant than normal and missing exons
4 and 17, respectively. These exon deletions introduce premature termination
codons predicted to truncate the proteins near the N and C termini,
respectively. No mutations in the genes encoding Picalm, clathrin, or
components of the adaptor protein complex 2 (AP2) have been previously
described in which the suite of disorders present in the

Picalm
fit1

mutant mice is apparent. These mutants thus
provide unique models for exploring how the endocytic function of mouse Picalm
and the transport processes mediated by clathrin and the AP2 complex
contribute to normal hematopoiesis, iron metabolism, and growth.
Lytic but not latent infection by Kaposi's sarcoma-associated herpesvirus requires host CSL protein, the mediator of Notch signaling
Authors: Liang Yuying , Ganem Don
Published: July 2003

Infection by Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a key
factor in the development of KS. Both latent and lytic KSHV infection is
observed in KS tumor cells, and both genetic programs contribute importantly
to KS pathogenesis. The viral replication and transcription activator (RTA)
protein is a transcription factor that controls the switch from latency to
lytic replication. We have previously shown that RTA can activate the
expression of several lytic viral genes in transfected cells by interaction
with recombination signal sequence-binding protein-Jκ (RBP-Jκ,
also called CSL), which in uninfected cells is a transcriptional repressor
that is the target of the Notch-signaling pathway. The recognition that many
KSHV lytic genes, including RTA itself, contain RBP-Jκ-binding sites
raised the possibility that RBP-Jκ-mediated repression may be central to
the establishment of latency. Here, we have tested this hypothesis by
examining KSHV infection of RBP-Jκ-null murine fibroblasts. Our results
show that KSHV latency is efficiently induced in such cells; however, the
reactivation of lytic gene expression, viral DNA replication, and the release
of progeny viruses are dramatically inhibited in the absence of RBP-Jκ.
RBP-Jκ-mediated repression is therefore not essential for establishment
of latent infection, but the RTA-mediated redirection of RBP-Jκ activity
from repression to activation is critical for lytic viral replication.
Angiotensin-(1–7) is an endogenous ligand for the G protein-coupled receptor Mas
Authors: Santos Robson A. S. , e Silva Ana C. Simoes , Maric Christine , Silva Denise M. R. , Machado Raquel Pillar , de Buhr Insa , Heringer-Walther Silvia , Pinheiro Sergio Veloso B. , Lopes Myriam Teresa , Bader Michael , Mendes Elizabeth P. , Lemos Virgina Soares , Campagnole-Santos Maria Jose , Schultheiss Heinz-Peter , Speth Robert , Walther Thomas
Published: July 2003

The renin–angiotensin system plays a critical role in blood pressure
control and body fluid and electrolyte homeostasis. Besides angiotensin (Ang)
II, other Ang peptides, such as Ang III [Ang-(2–8)], Ang IV
[Ang-(3–8)], and Ang-(1–7) may also have important biological
activities. Ang-(1–7) has become an angiotensin of interest in the past
few years, because its cardiovascular and baroreflex actions counteract those
of Ang II. Unique angiotensin-binding sites specific for this heptapeptide and
studies with a selective Ang-(1–7) antagonist indicated the existence of
a distinct Ang-(1–7) receptor. We demonstrate that genetic deletion of
the G protein-coupled receptor encoded by the
Mas
protooncogene
abolishes the binding of Ang-(1–7) to mouse kidneys. Accordingly,
Mas
-deficient mice completely lack the antidiuretic action of
Ang-(1–7) after an acute water load. Ang-(1–7) binds to
Mas
-transfected cells and elicits arachidonic acid release.
Furthermore, Mas-deficient aortas lose their Ang-(1–7)-induced
relaxation response. Collectively, these findings identify Mas as a functional
receptor for Ang-(1–7) and provide a clear molecular basis for the
physiological actions of this biologically active peptide.
Regulatory CD8+T cells fine-tune the myelin basic protein-reactive T cell receptor Vβ repertoire during experimental autoimmune encephalomyelitis
Authors: Jiang H. , Curran S. , Ruiz-Vazquez E. , Liang B. , Winchester R. , Chess L.
Published: July 2003

A significant number of self-reactive T cell clones escape thymic negative selection and are released into the periphery, where some are potentially pathogenic. The clonal expansion of self-reactive T cells is known to be limited during initial antigen encounter by apoptotic or anergic mechanisms, regulatory CD4+T cells, and cytokines. Here we report that superimposed on these mechanisms, during the evolution of autoimmunity in experimental autoimmune encephalomyelitis (EAE), CD8+T cells are induced, which fine-tune the peripheral self-reactive T cell receptor (TCR) repertoire. We assayed the myelin basic protein-reactive TCR repertoire in naive, EAE-recovered mice as well as EAE-recovered mice depleted of CD8+T cells by TCRVβ surface expression, complementarity-determining region 3 length distribution, and complementarity-determining region 3 sequencing analysis. In EAE-recovered mice, certain myelin basic protein-reactive CD4+Vβ8.2+clones are significantly decreased and this decrease is not observed if CD8+T cells were depleted from these mice. The clones that persist in CD8+T cell-intact mice are highly diverse in contrast to the clones expanded in CD8+T cell-depleted mice, which are dominated by the significant outgrowth of a few clones. Importantly, the T cell clones that expand in the absence of CD8+T cell control are enriched in potentially pathogenic self-reactive T cell clones capable of inducing EAEin vivo.
Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules
Authors: Christensen Erik I. , Devuyst Olivier , Dom Geneviève , Nielsen Rikke , Van Der Smissen Patrick , Verroust Pierre , Leruth Michèle , Guggino William B. , Courtoy Pierre J.
Published: July 2003

Loss of the renal endosome-associated chloride channel, ClC-5, in Dent's
disease and knockout (KO) mice strongly inhibits endocytosis of filtered
proteins by kidney proximal tubular cells (PTC). The underlying mechanism
remains unknown. We therefore tested whether this endocytic failure could
primarily reflect a loss of reabsorption by the multiligand receptors,
megalin, and cubilin, caused by a trafficking defect. Impaired protein
endocytosis in PTC of ClC-5 KO mice was demonstrated by (
i
) a major
decreased uptake of injected
125
I-β
2
-microglobulin,
but not of the fluid-phase tracer, FITC-dextran, (
ii
) reduced
labeling of endosomes by injected peroxidase and for the endogenous
megalin/cubilin ligands, vitamin D- and retinol-binding proteins, and
(
iii
) urinary appearance of low-molecular-weight proteins and the
selective cubilin ligand, transferrin. Contrasting with preserved mRNA levels,
megalin and cubilin abundance was significantly decreased in kidney extracts
of KO mice. Percoll gradients resolving early and late endosomes (Rab5a,
Rab7), brush border (villin, aminopeptidase M), and a dense peak comprising
lysosomes (acid hydrolases) showed a disappearance of the brush border
component for megalin and cubilin in KO mice. Quantitative ultrastructural
immunogold labeling confirmed the overall decrease of megalin and cubilin in
PTC and their selective loss at the brush border. In contrast, total contents
of the rate-limiting endocytic catalysts, Rab5a and Rab7, were unaffected.
Thus, impaired protein endocytosis caused by invalidation of ClC-5 primarily
reflects a trafficking defect of megalin and cubilin in PTC.
TNF-αneutralization ameliorates the severity of murine Crohn's-like ileitis by abrogation of intestinal epithelial cell apoptosis
Authors: Marini Marco , Bamias Giorgos , Rivera-Nieves Jesús , Moskaluk Christopher A. , Hoang Sharon B. , Ross William G. , Pizarro Theresa T. , Cominelli Fabio
Published: July 2003

Tumor necrosis factor α (TNF-α) is an important mediator of programmed cell death, and TNF-α blockade significantly improves disease severity in several inflammatory conditions, including Crohn's disease (CD), one of the idiopathic inflammatory bowel diseases. However, the precise mechanism(s) of action of anti-TNF-α therapy in CD remains poorly understood. SAMP1/YitFc mice develop a spontaneous ileitis with similarities to human CD in regard to histological features as well as response to conventional treatments. In this report, we tested the novel hypothesis that the beneficial effects of anti-TNF-α therapy in CD are mediated by a mechanism that involves down-regulation of intestinal epithelial cell (IEC) apoptosis. Similar to the efficacy of monoclonal anti-TNF-α antibodies in human CD, a single injection of a chimeric anti-murine TNF-α antibody into SAMP1/YitFc mice resulted in a marked suppression of intestinal inflammation and epithelial cell damage compared with mice injected with an isotype control antibody. These effects were associated with a significant reduction in apoptosis of freshly isolated IEC as assessed by propidium iodide staining and DNA laddering. In contrast, an increase in lamina propria mononuclear cell apoptosis was observed in anti-TNF-α-treated mice compared with control. These results were confirmedin vivoby using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-assay. In addition, neutralization of TNF-α reduced membrane bound FAS/CD95 expression in IEC from SAMP1/YitFc mice compared with control antibody. These data demonstrate a novel mechanism of action of anti-TNF-α therapy that involves homeostatic regulation of mucosal cell apoptosis, which results in the net decrease of chronic inflammation typically found in CD.
A homologue of elongation factor 1γ regulates methionine sulfoxide reductase A gene expression in
Saccharomyces cerevisiae
Authors: Hanbauer Ingeborg , Boja Emily S. , Moskovitz Jackob
Published: July 2003

Methionine sulfoxide reductase A (MsrA) maintains the function of many
proteins by reversing oxidation of methionine residues. Lack of this repair
mechanism very likely increases aging-related disease susceptibility. In
Saccharomyces cerevisiae
, disruption of the
msrA
gene
increases free and protein-bound methionine sulfoxide and decreases cell
viability. Although the underlying mechanisms in the induction of the
msrA
gene are still unknown, a transcriptional regulation may be
involved. Hence, a search of nuclear proteins regulating the
msrA
gene is a major target of the experiments reported in this article. Using
protein purification combined with MS, we discovered that calcium
phospholipid-binding protein (CPBP), a homologue of elongation
factor-1γ, is a component of a complex that binds to the
msrA
promoter. By measuring CPBP cooperative binding to the
msrA
promoter,
we have mapped the CPBP binding site to a 39-bp sequence at the 3′ end
of the promoter. In a mutant yeast strain lacking the CPBP-encoding gene, the
ability to overexpress
msrA
mRNA and MsrA protein was impaired and
MsrA catalytic activity was greatly reduced, suggesting that CPBP may enhance
msrA
gene expression.
Evolutionary genomics of inversions in
Drosophila pseudoobscura
: Evidence for epistasis
Authors: Schaeffer Stephen W. , Goetting-Minesky M. Paula , Kovacevic Miro , Peoples John R. , Graybill Jennifer L. , Miller Jonathan M. , Kim Kyungsun , Nelson Julie G. , Anderson Wyatt W.
Published: July 2003

Drosophila pseudoobscura
harbors a rich polymorphism for
paracentric inversions on the third chromosome, and the clines in the
inversion frequencies across the southwestern United States indicate that
strong natural selection operates on them. Isogenic inversion strains were
made from isofemale lines collected from four localities, and eight molecular
markers were mapped on the third chromosome. Nucleotide diversity was measured
for these loci and formed the basis of an evolutionary genomic analysis. The
loci were differentiated among inversions. The inversions did not show
significant differences among populations, however, likely the result of
extensive gene flow among populations. Some loci had significant reductions in
nucleotide diversity within inversions compared with interspecies divergence,
suggesting that these loci are near inversion breakpoints or are near targets
of directional selection. Linkage disequilibrium (LD) levels tended to
decrease with distance between loci, indicating that some genetic exchange
occurs among gene arrangements despite the presence of inversions. In some
cases, however, adjacent genes had low levels of interlocus LD and loosely
linked genes had high levels of interlocus LD, suggesting strong epistatic
selection. Our results support the hypothesis that the inversions of
D.
pseudoobscura
have emerged as suppressors of recombination to maintain
positive epistatic relationships among loci within gene arrangements that
developed as the species adapted to a heterogeneous environment.
Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects
Authors: Ortiz Luis A. , Gambelli Frederica , McBride Christine , Gaupp Dina , Baddoo Melody , Kaminski Naftali , Phinney Donald G.
Published: July 2003

Previously we described a reliable method based on immunodepletion for
isolating mesenchymal stem cells (MSCs) from murine bone marrow and showed
that, after intracranial transplantation, the cells migrated throughout
forebrain and cerebellum and adopted neural cell fates. Here we systemically
administered MSCs purified by immunodepletion from male bleomycin
(BLM)-resistant BALB/c mice into female BLM-sensitive C57BL/6 recipients and
quantified engraftment levels in lung by real-time PCR. Male DNA accounted for
2.21 × 10
-5
% of the total lung DNA in control-treated mice
but was increased 23-fold (
P
= 0.05) in animals exposed to BLM before
MSC transplantation. Fluorescence
in situ
hybridization revealed that
engrafted male cells were localized to areas of BLM-induced injury and
exhibited an epithelium-like morphology. Moreover, purification of type II
epithelial cells from the lungs of transplant recipients resulted in a 3-fold
enrichment of male, donor-derived cells as compared with whole lung tissue.
MSC administration immediately after exposure to BLM also significantly
reduced the degree of BLM-induced inflammation and collagen deposition within
lung tissue. Collectively, these studies demonstrate that murine MSCs home to
lung in response to injury, adopt an epithelium-like phenotype, and reduce
inflammation and collagen deposition in lung tissue of mice challenged with
BLM.
Promotion of Gαi3 subunit down-regulation by GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP
Authors: Fischer Thierry , De Vries Luc , Meerloo Timo , Farquhar Marilyn Gist
Published: July 2003

We have isolated an RGS-GAIP interacting protein that links RGS proteins to
protein degradation. GIPN (GAIP interacting protein N terminus) is a 38-kDa
protein with an N-terminal leucine-rich region, a central RING finger-like
domain, and a putative C-terminal transmembrane domain. GIPN binds exclusively
to RGS proteins of subfamily A, RGS-GAIP, RGSZ1, and RGSZ2. The N-terminal
leucine-rich region of GIPN interacts with the cysteine-rich motif of
RGS-GAIP. GIPN mRNA is ubiquitously expressed, and GIPN is found on the plasma
membrane of transfected HEK293 cells. Endogenous GIPN is concentrated along
the basolateral plasma membrane of proximal and distal tubules in rat kidney,
where many G protein-coupled receptors and some G proteins are also located.
Two immunoreactive species are found in rat kidney, a 38-kDa cytosolic form
and an ≈94-kDa membrane form. GIPN shows Zn
2+
- and
E1/E2-dependent autoubiquitination
in vitro
, suggesting that it has
E3 ubiquitin ligase activity. Overexpression of GIPN stimulates
proteasome-dependent reduction of endogenous Gαi3 in HEK293 cells and
reduces the half-life of overexpressed Gαi3-YFP. Thus, our findings
suggest that GIPN is involved in the degradation of Gαi3 subunits via
the proteasome pathway. RGS-GAIP functions as a bifunctional adaptor that
binds to Gα subunits through its RGS domain and to GIPN through its
cysteine string motif.
Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis
Authors: Whittington Douglas A. , Rusche Kristin M. , Shin Hyunshun , Fierke Carol A. , Christianson David W.
Published: July 2003

The outer leaflet of the outer membrane of the Gram-negative bacterium
serves as a permeability barrier and is composed of lipopolysaccharide, also
known as endotoxin. The membrane anchor of lipopolysaccharide is lipid A, the
biosynthesis of which is essential for cell viability. The first committed
step in lipid A biosynthesis is catalyzed by
UDP-(3-
O
-(
R
-3-hydroxymyristoyl))-
N
-acetylglucosamine
deacetylase (LpxC), a zinc-dependent deacetylase. Here we report the crystal
structure of LpxC from
Aquifex aeolicus
, which reveals a new
α+β fold reflecting primordial gene duplication and fusion, as well
as a new zinc-binding motif. The catalytic zinc ion resides at the base of an
active-site cleft and adjacent to a hydrophobic tunnel occupied by a fatty
acid. This tunnel accounts for the specificity of LpxC toward substrates and
inhibitors bearing appropriately positioned 3-
O
-fatty acid
substituents. Notably, simple inhibitors designed to target interactions in
the hydrophobic tunnel bind with micromolar affinity, thereby representing a
step toward the structure-based design of a potent, broad-spectrum
antibacterial drug.