bluuebunny/crossref_metadata_2025 huggingface dataset:
Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection
Authors: Hurt K. Joseph , Musicki Biljana , Palese Michael A. , Crone Julie K. , Becker Robyn E. , Moriarity John L. , Snyder Solomon H. , Burnett Arthur L.
Published: July 2002

In the penis, nitric oxide (NO) can be formed by both neuronal NO synthase and endothelial NOS (eNOS). eNOS is activated by viscous drag/shear stress in blood vessels to produce NO continuously, a process mediated by the phosphatidylinositol 3-kinase (PI3kinase)/Akt pathway. Here we show that PI3-kinase/Akt physiologically mediates erection. Both electrical stimulation of the cavernous nerve and direct intracavernosal injection of the vasorelaxant drug papaverine cause rapid increases in phosphorylated (activated) Akt and eNOS. Phosphorylation is diminished by wortmannin and LY294002, inhibitors of PI3-kinase, the upstream activator of Akt. The two drugs also reduce erection. Penile erection elicited by papaverine is reduced profoundly in mice with targeted deletion of eNOS. Our findings support a model in which rapid, brief activation of neuronal NOS initiates the erectile process, whereas PI3-kinase/Akt-dependent phosphorylation and activation of eNOS leads to sustained NO production and maximal erection.
Peyer's patches are required for oral tolerance to proteins
Authors: Fujihashi Kohtaro , Dohi Taeko , Rennert Paul D. , Yamamoto Masafumi , Koga Toshiya , Kiyono Hiroshi , McGhee Jerry R.
Published: July 2002

To clarify the role of Peyer's patches in oral tolerance induction, BALB/c mice were treatedin uterowith lymphotoxin β-receptor Ig fusion protein to generate mice lacking Peyer's patches. When these Peyer's patch-null mice were fed 25 mg of ovalbumin (OVA) before systemic immunization, OVA-specific IgG Ab responses in serum and spleen were seen, in marked contrast to low responses in OVA-fed normal mice. Further, high T-cell-proliferative- and delayed-type hypersensitivity responses were seen in Peyer's patch-null mice given oral OVA before systemic challenge. Higher levels of CD4+T-cell-derived IFN-γ, IL-4, IL-5, and IL-10 syntheses were noted in Peyer's patch-null mice fed OVA, whereas OVA-fed normal mice had suppressed cytokine levels. In contrast, oral administration of trinitrobenzene sulfonic acid (TNBS) to Peyer's patch-null mice resulted in reduced TNBS-specific serum Abs and splenic B cell antitrinitrophenyl Ab-forming cell responses after skin painting with picryl chloride. Further, when delayed-type hypersensitivity and splenic T cell proliferative responses were examined, Peyer's patch-null mice fed TNBS were unresponsive to hapten. Peyer's patch-null mice fed trinitrophenyl-OVA failed to induce systemic unresponsiveness to hapten or protein. These findings show that organized Peyer's patches are required for oral tolerance to proteins, whereas haptens elicit systemic unresponsiveness via the intestinal epithelial cell barrier.
Simulating materials failure by using up to one billion atoms and the world's fastest computer: Work-hardening
Authors: Abraham Farid F. , Walkup Robert , Gao Huajian , Duchaineau Mark , Diaz De La Rubia Tomas , Seager Mark
Published: July 2002

We describe the second of two large-scale atomic simulation projects on materials failure performed on the 12-teraflop ASCI (Accelerated Strategic Computing Initiative) White computer at the Lawrence Livermore National Laboratory. This investigation simulates ductile failure by using more than one billion atoms where the true complexity of the creation and interaction of hundreds of dislocations are revealed.
Three-dimensional image reconstruction of dephosphorylated smooth muscle heavy meromyosin reveals asymmetry in the interaction between myosin heads and placement of subfragment 2
Authors: Wendt Thomas , Taylor Dianne , Trybus Kathleen M. , Taylor Kenneth
Published: July 2002

Regulation of the actin-activated ATPase of smooth muscle myosin II
is known to involve an interaction between the two heads that is
controlled by phosphorylation of the regulatory light chain. However,
the three-dimensional structure of this inactivated form has been
unknown. We have used a lipid monolayer to obtain two-dimensional
crystalline arrays of the unphosphorylated inactive form of smooth
muscle heavy meromyosin suitable for structural studies by electron
cryomicroscopy of unstained, frozen-hydrated specimens. The
three-dimensional structure reveals an asymmetric interaction
between the two myosin heads. The ATPase activity of one head
is sterically “blocked” because part of its actin-binding
interface is positioned onto the converter domain of the second head.
ATPase activity of the second head, which can bind actin, appears to be
inhibited through stabilization of converter domain movements needed to
release phosphate and achieve strong actin binding. When the
subfragment 2 domain of heavy meromyosin is oriented as it would be in
an actomyosin filament lattice, the position of the heads is very
different from that needed to bind actin, suggesting an additional
contribution to ATPase inhibition
in situ
.
Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion
Authors: Royaux Ines E. , Wall Susan M. , Karniski Lawrence P. , Everett Lorraine A. , Suzuki Koichi , Knepper Mark A. , Green Eric D.
Published: July 2002

Pendrin is an anion transporter encoded by the
PDS
/
Pds
gene. In humans, mutations in
PDS
cause the genetic disorder Pendred syndrome, which
is associated with deafness and goiter. Previous studies have shown
that this gene has a relatively restricted pattern of expression, with
PDS
/
Pds
mRNA detected only in the
thyroid, inner ear, and kidney. The present study examined the
distribution and function of pendrin in the mammalian kidney.
Immunolocalization studies were performed using anti-pendrin polyclonal
and monoclonal antibodies. Labeling was detected on the apical surface
of a subpopulation of cells within the cortical collecting ducts (CCDs)
that also express the H
+
-ATPase but not aquaporin-2,
indicating that pendrin is present in intercalated cells of the CCD.
Furthermore, pendrin was detected exclusively within the subpopulation
of intercalated cells that express the H
+
-ATPase but not
the anion exchanger 1 (AE1) and that are thought to mediate bicarbonate
secretion. The same distribution of pendrin was observed in mouse, rat,
and human kidney. However, pendrin was not detected in kidneys from a
Pds
-knockout mouse. Perfused CCD tubules isolated from
alkali-loaded wild-type mice secreted bicarbonate, whereas tubules from
alkali-loaded
Pds
-knockout mice failed to secrete
bicarbonate. Together, these studies indicate that pendrin is an apical
anion transporter in intercalated cells of CCDs and has an essential
role in renal bicarbonate secretion.
Activation of a protease cascade involved in patterning the
Drosophila
embryo
Authors: LeMosy Ellen K. , Tan Yue-Qing , Hashimoto Carl
Published: July 2002

Dorsoventral patterning of the
Drosophila
embryo is
initiated by a ventralizing signal. Production of this signal requires
the serine proteases Gastrulation Defective (GD), Snake, and Easter,
which genetic studies suggest act sequentially in a cascade that is
activated locally in response to a ventral cue provided by the
pipe
gene. Here, we demonstrate biochemically that GD
activates Snake, which in turn activates Easter. We also provide
evidence that GD zymogen cleavage is important for triggering this
cascade but is not spatially localized by
pipe
. Our
results suggest that a broadly, rather than locally, activated protease
cascade produces the ventralizing signal, so a distinct downstream step
in this cascade must be spatially regulated to restrict signaling to
the ventral side of the embryo.
Epstein–Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: Protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors
Authors: Higuchi Masaya , Izumi Kenneth M. , Kieff Elliott
Published: July 2002

Epstein–Barr virus encodes integral membrane proteins LMP1 and LMP2A in transformed lymphoblastoid cell lines. We now find that LMP1 associates with the cell cytoskeleton through a tumor necrosis factor receptor-associated factor-interacting domain, most likely mediated by tumor necrosis factor receptor-associated factor 3. LMP1 is palmitoylated, and the transmembrane domains associate with lipid rafts. Mutation of LMP1 cysteine-78 abrogates palmitoylation but does not affect raft association or NF-κB or c-Jun N-terminal kinase activation. LMP2A also associates with rafts and is palmitoylated but does not associate with the cell cytoskeleton. The associations of LMP1 and LMP2A with rafts and of LMP1 with the cell cytoskeleton are likely to effect interactions with cell proteins involved in shape, motility, signal transduction, growth, and survival.
Recognition of a virus-encoded ligand by a natural killer cell activation receptor
Authors: Smith Hamish R. C. , Heusel Jonathan W. , Mehta Indira K. , Kim Sungjin , Dorner Brigitte G. , Naidenko Olga V. , Iizuka Koho , Furukawa Hiroshi , Beckman Diana L. , Pingel Jeanette T. , Scalzo Anthony A. , Fremont Daved H. , Yokoyama Wayne M.
Published: July 2002

Natural killer (NK) cells express inhibitory and activation receptors that recognize MHC class I-like molecules on target cells. These receptors may be involved in the critical role of NK cells in controlling initial phases of certain viral infections. Indeed, the Ly49H NK cell activation receptor confersin vivogenetic resistance to murine cytomegalovirus (MCMV) infections, but its ligand was previously unknown. Herein, we use heterologous reporter cells to demonstrate that Ly49H recognizes MCMV-infected cells and a ligand encoded by MCMV itself. Exploiting a bioinformatics approach to the MCMV genome, we find at least 11 ORFs for molecules with previously unrecognized features of predicted MHC-like folds and limited MHC sequence homology. We identify one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-mediated cytotoxicity, and cytokine and chemokine production by freshly isolated NK cells. We hypothesize that the other ORFs with predicted MHC-like folds may be involved in immune evasion or interactions with other NK cell receptors.
Umbrellas and flagships: Efficient conservation surrogates or expensive mistakes?
Authors: Andelman Sandy J. , Fagan William F.
Published: July 2002

The use of umbrella and flagship species as surrogates for regional biota whose spatial distributions are poorly known is a popular conservation strategy. Yet many assumptions underlying the choice of surrogate species remain untested. By using biodiversity databases containing spatial incidence data for species of concern for (
i
) the southern California coastal sage scrub habitat, (
ii
) the Columbia Plateau ecoregion, and (
iii
) the continental United States, we evaluate the potential effectiveness of a range of conservation surrogate schemes (e.g., big carnivores, charismatic species, keystone species, wide-ranging species), asking how many species potentially are protected by each scheme and at what cost in each habitat area. For all three databases, we find that none of the surrogate schemes we evaluated performs significantly better than do a comparable number of species randomly selected from the database. Although some surrogate species may have considerable publicity value, based on the databases we analyzed, representing diverse taxa on three different geographic scales, we find that the utility of umbrella and flagship species as surrogates for regional biodiversity may be limited.
Designing surfaces that kill bacteria on contact
Authors: Tiller Joerg C. , Liao Chun-Jen , Lewis Kim , Klibanov Alexander M.
Published: July 2002

Poly(4-vinyl-
N
-alkylpyridinium bromide) was
covalently attached to glass slides to create a surface that kills
airborne bacteria on contact. The antibacterial properties were
assessed by spraying aqueous suspensions of bacterial cells on the
surface, followed by air drying and counting the number of cells
remaining viable (i.e., capable of growing colonies). Amino glass
slides were acylated with acryloyl chloride, copolymerized with
4-vinylpyridine, and
N
-alkylated with different alkyl
bromides (from propyl to hexadecyl). The resultant surfaces, depending
on the alkyl group, were able to kill up to 94 ± 4% of
Staphylococcus aureus
cells sprayed on them. A surface
alternatively created by attaching poly(4-vinylpyridine) to a glass
slide and alkylating it with hexyl bromide killed 94 ± 3% of the
deposited
S. aureus
cells. On surfaces modified with
N
-hexylated poly(4-vinylpyridine), the numbers of viable
cells of another Gram-positive bacterium,
Staphylococcus
epidermidis
, as well as of the Gram-negative bacteria
Pseudomonas aeruginosa
and
Escherichia
coli
, dropped more than 100-fold compared with the original
amino glass. In contrast, the number of viable bacterial cells did not
decline significantly after spraying on such common materials as
ceramics, plastics, metals, and wood.
A rice spotted leaf gene,
Spl7
, encodes a heat stress transcription factor protein
Authors: Yamanouchi Utako , Yano Masahiro , Lin Hongxuan , Ashikari Motoyuki , Yamada Kyoji
Published: July 2002

A rice spotted leaf (lesion-mimic) gene,
Spl7
, was identified by map-based cloning. High-resolution mapping with cleaved amplified polymorphic sequence markers enabled us to define a genomic region of 3 kb as a candidate for
Spl7.
We found one ORF that showed high similarity to a heat stress transcription factor (HSF). Transgenic analysis verified the function of the candidate gene for
Spl7
: leaf spot development was suppressed in
spl7
mutants with a wild-type
Spl7
transgene. Thus, we conclude that
Spl7
encodes the HSF protein. The transcript of
spl7
was observed in mutant plants. The levels of mRNAs (
Spl7
in wild type and
spl7
in mutant) increased under heat stress. Sequence analysis revealed only one base substitution in the HSF DNA-binding domain of the mutant allele, causing a change from tryptophan to cysteine.
Indium-cadmium-oxide films having exceptional electrical conductivity and optical transparency: Clues for optimizing transparent conductors
Authors: Wang A. , Babcock J. R. , Edleman N. L. , Metz A. W. , Lane M. A. , Asahi R. , Dravid V. P. , Kannewurf C. R. , Freeman A. J. , Marks T. J.
Published: July 2002

Materials with high electrical conductivity and optical transparency are needed for future flat panel display, solar energy, and other opto-electronic technologies. In

x

Cd
1-

x

O films having a simple cubic microstructure have been grown on amorphous glass substrates by a straightforward chemical vapor deposition process. The
x
= 0.05 film conductivity of 17,000 S/cm, carrier mobility of 70 cm
2
/Vs, and visible region optical transparency window considerably exceed the corresponding parameters for commercial indium-tin oxide.
Ab initio
electronic structure calculations reveal small conduction electron effective masses, a dramatic shift of the CdO band gap with doping, and a conduction band hybridization gap caused by extensive Cd 5s + In 5s mixing.
Regulation of SOS1, a plasma membrane Na
+
/H
+
exchanger in
Arabidopsis
thaliana
, by SOS2 and SOS3
Authors: Qiu Quan-Sheng , Guo Yan , Dietrich Margaret A. , Schumaker Karen S. , Zhu Jian-Kang
Published: July 2002

Maintaining low levels of sodium ions in the cell cytosol is critical for plant growth and development. Biochemical studies suggest that Na
+
/H
+
exchangers in the plasma membrane of plant cells contribute to cellular sodium homeostasis by transporting sodium ions out of the cell; however, these exchangers have not been identified at the molecular level. Genetic analysis has linked components of the
s
alt
o
verly
s
ensitive pathway (
SOS1
–
3
) to salt tolerance in
Arabidopsis thaliana
. The predicted SOS1 protein sequence and comparisons of sodium ion accumulation in wild-type and
sos1
plants suggest that SOS1 is involved directly in the transport of sodium ions across the plasma membrane. To demonstrate the transport capability of SOS1, we studied Na
+
/H
+
-exchange activity in wild-type and
sos
plants using highly purified plasma membrane vesicles. The results showed that plasma membrane Na
+
/H
+
-exchange activity was present in wild-type plants treated with 250 mM NaCl, but this transport activity was reduced by 80% in similarly treated
sos1
plants.
In vitro
addition of activated SOS2 protein (a protein kinase) increased Na
+
/H
+
-exchange activity in salt-treated wild-type plants 2-fold relative to transport without added protein. However, the addition of activated SOS2 did not have any stimulatory effect on the exchange activity in
sos1
plants. Although vesicles of
sos2
and
sos3
plants had reduced plasma membrane Na
+
/H
+
-exchange activity, transport activity in both increased with the addition of activated SOS2 protein. These results demonstrate that SOS1 contributes to plasma membrane Na
+
/H
+
exchange and that SOS2 and SOS3 regulate SOS1 transport activity.
B cell receptor expression level determines the fate of developing B lymphocytes: Receptor editing versus selection
Authors: Kouskoff Valerie , Lacaud Georges , Pape Kathryn , Retter Marc , Nemazee David
Published: July 2002

During B lymphocyte development, antibody genes are assembled by DNA recombination. Successful cell surface expression of IgM promotes developmental progression. However, when antigen receptors bind autoantigen, development is blocked and ongoing antibody gene recombination occurs, which often alters antibody specificity in a process called receptor editing. We demonstrate here a significant role of developmental block and receptor editing in B cell receptor quality control. During development a functional, non-self-reactive receptor undergoes receptor editing if its expression is below a certain threshold. Doubling the receptor gene dose promotes development in the absence of autoantigen, but allows editing when autoantigen is present. Thus, both underexpressed and harmful B cell receptors can undergo correction by receptor editing.
The C-terminal domain-phosphorylated IIO form of RNA polymerase II is associated with the transcription repressor NC2 (Dr1/DRAP1) and is required for transcription activation in human nuclear extracts
Authors: Castaño Enrique , Gross Petra , Wang Zhengxin , Roeder Robert G. , Oelgeschläger Thomas
Published: July 2002

Activation of class II gene transcription may involve alleviation of transcription repression as well as stimulation of the assembly and function of the general RNA polymerase (RNAP) II transcription machinery. Here, we investigated whether activator-reversible transcription repression by NC2 (Dr1/DRAP1) contributes to maximum induction levels in unfractionated HeLa nuclear extracts. Surprisingly, we found that depletion of NC2 does not significantly affect basal transcription, but dramatically reduces activated transcription. Immunoblot analyses revealed that the loss of activator function coincides with selective removal of the C-terminal domain (CTD)-hyperphosphorylated RNAP IIO along with NC2. Coimmunoprecipitation experiments with purified factors confirmed that NC2 interacts with RNAP IIO, but not with the unphosphorylated or hypophosphorylated RNAP IIA or CTD-less RNAP IIB forms. Finally, we demonstrate that, in contrast to previously published observations in cell-free systems reconstituted with purified factors, only the CTD-phosphorylated form of RNAP II can mediate activator function in the context of unfractionated HeLa nuclear extracts. These findings reveal an unexpected link between NC2 and transcription activation and suggest that regulation of RNAP II transcription through reversible CTD phosphorylation might be more complex than previously proposed.
Trace amines: Identification of a family of mammalian G protein-coupled receptors
Authors: Borowsky Beth , Adham Nika , Jones Kenneth A. , Raddatz Rita , Artymyshyn Roman , Ogozalek Kristine L. , Durkin Margaret M. , Lakhlani Parul P. , Bonini James A. , Pathirana Sudam , Boyle Noel , Pu Xiaosui , Kouranova Evguenia , Lichtblau Harvey , Ochoa F. Yulina , Branchek Theresa A. , Gerald Christophe
Published: July 2002

Tyramine, β-phenylethylamine, tryptamine, and octopamine are biogenic amines present in trace levels in mammalian nervous systems. Although some “trace amines” have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Using a degenerate PCR approach, we have identified 15 G protein-coupled receptors (GPCR) from human and rodent tissues. Together with the orphan receptor PNR, these receptors form a subfamily of rhodopsin GPCRs distinct from, but related to the classical biogenic amine receptors. We have demonstrated that two of these receptors bind and/or are activated by trace amines. The cloning of mammalian GPCRs for trace amines supports a role for trace amines as neurotransmitters in vertebrates. Three of the four human receptors from this family are present in the amygdala, possibly linking trace amine receptors to affective disorders. The identification of this family of receptors should rekindle the investigation of the roles of trace amines in mammalian nervous systems and may potentially lead to the development of novel therapeutics for a variety of indications.
Histone deacetylase-dependent transcriptional repression by pRB in yeast occurs independently of interaction through the LXCXE binding cleft
Authors: Kennedy Brian K. , Liu Oliver W. , Dick Fred A. , Dyson Nick , Harlow Ed , Vidal Marc
Published: July 2002

We have developed a yeast model system to address transcriptional repression by the retinoblastoma protein (pRB). When fused to the DNA-binding domain of Gal4p (DB-pRB), pRB can repress transcription of reporter genes containing Gal4p binding sites; the histone deacetylase activity encoded by yeast
RPD3
is required for DB-pRB repression. Mutation of the LXCXE binding cleft in pRB, a region reported to be required for histone deacetylase recruitment, does not interfere with pRB-mediated repression. From these findings based on yeast experiments, we surmise that the small pocket region of pRB must contain an additional domain that confers histone deacetylase-dependent transcriptional repression. This hypothesis was verified by experiments examining pRB-dependent histone deacetylase association in mammalian cells. In addition to
RPD3
, repression by pRB in yeast requires
MSI1
, an ortholog of RbAp48, but not
SIN3
or
SAP30
. By comparing the genetic requirements of DB-pRB repression in yeast to those of other DB-repressor fusions, we can suggest a mechanism by which pRB recruits histone deacetylase activity.
An antisense approach to phenotype-based gene cloning in
Tetrahymena
Authors: Chilcoat N. Doane , Elde Nels C. , Turkewitz Aaron P.
Published: July 2002

We report a pioneering approach using
Tetrahymena thermophila
that permits rapid identification of genes based on their null or hypomorphic phenotypes. This technique involves cell transformation with a library of plasmids that encode 26S ribosomal subunits containing short insertions. The insertions correspond to antisense sequences for a large number of genes. The majority of cells each acquires a single antisense sequence, which silences a single genomic locus. Because the insertion site within the ribosomal sequence is known, the silenced gene is easily amplified. We demonstrate that this approach can be used to identify genes required for dense core granule exocytosis.
Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose
Authors: Carrard Géraldine , Koivula Anu , Söderlund Hans , Béguin Pierre
Published: July 2002

The cohesin-dockerin interaction in
Clostridium thermocellum
cellulosome mediates the tight binding of cellulolytic enzymes to the cellulosome-integrating protein CipA. Here, this interaction was used to study the effect of different cellulose-binding domains (CBDs) on the enzymatic activity of
C. thermocellum
endoglucanase CelD (1,4-β-
d
endoglucanase, EC
3.2.1.4
) toward various cellulosic substrates. The seventh cohesin domain of CipA was fused to CBDs originating from the
Trichoderma reesei
cellobiohydrolases I and II (CBD
CBH1
and CBD
CBH2
) (1,4-β-
d
glucan-cellobiohydrolase, EC
3.2.1.91
), from the
Cellulomonas fimi
xylanase/exoglucanase Cex (CBD
Cex
) (β-1,4-
d
glucanase, EC
3.2.1.8
), and from
C. thermocellum
CipA (CBD
CipA
). The CBD-cohesin hybrids interacted with the dockerin domain of CelD, leading to the formation of CelD-CBD complexes. Each of the CBDs increased the fraction of cellulose accessible to hydrolysis by CelD in the order CBD
CBH1
< CBD
CBH2
≈ CBD
Cex
< CBD
CipA
. In all cases, the extent of hydrolysis was limited by the disappearance of sites accessible to CelD. Addition of a batch of fresh cellulose after completion of the reaction resulted in a new burst of activity, proving the reversible binding of the intact complexes despite the apparent binding irreversibility of some CBDs. Furthermore, burst of activity also was observed upon adding new batches of CelD–CBD complexes that contained a CBD differing from the first one. This complementation between different CBDs suggests that the sites made available for hydrolysis by each of the CBDs are at least partially nonoverlapping. The only exception was CBD
CipA
, whose sites appeared to overlap all of the other sites.
Binding site of brefeldin A at the interface between the small G protein ADP-ribosylation factor 1 (ARF1) and the nucleotide-exchange factor Sec7 domain
Authors: Robineau Sylviane , Chabre Marc , Antonny Bruno
Published: July 2002

Sec7 domains (Sec7d) catalyze the exchange of guanine nucleotide on ARFs. Recent studies indicated that brefeldin A (BFA) inhibits Sec7d-catalyzed nucleotide exchange on ARF1 in an uncompetitive manner by trapping an early intermediate of the reaction: a complex between GDP-bound ARF1 and Sec7d. Using
3
H-labeled BFA, we show that BFA binds to neither isolated Sec7d nor isolated ARF1–GDP, but binds to the transitory Sec7d–ARF1–GDP complex and stabilizes it. Two pairs of residues at positions 190–191 and 198–208 (Arno numbering) in Sec7d contribute equally to the stability of BFA binding, which is also sensitive to mutation of H80 in ARF1. The catalytic glutamic (E156) residue of Sec7d is not necessary for BFA binding. In contrast, BFA does not bind to the intermediate catalytic complex between nucleotide-free ARF1 and Sec7d. These results suggest that, on initial docking steps between ARF1–GDP and Sec7d, BFA inserts like a wedge between the switch II region of ARF1–GDP and a surface encompassing residues 190–208, at the border of the characteristic hydrophobic groove of Sec7d. Bound BFA would prevent the switch regions of ARF1–GDP from reorganizing and forming tighter contacts with Sec7d and thereby would maintain the bound GDP of ARF1 at a distance from the catalytic glutamic finger of Sec7d.
Assessment and discrimination of odor stimuli in rat olfactory bulb by dynamic functional MRI
Authors: Xu Fuqiang , Kida Ikuhiro , Hyder Fahmeed , Shulman Robert G.
Published: July 2002

Dynamic blood oxygenation level-dependent functional MRI was applied at 7 T in the rat olfactory bulb (OB) with pulsed delivery of iso-amyl acetate (IAA) and limonene. Acquisition times for single-slice and whole OB data were 8 and 32 s, respectively, with spatial resolution of 220 × 220 × 250 μm. On an intrasubject basis, short IAA exposures of 0.6 min separated by 3.5-min intervals induced reproducible spatial activity patterns (SAPs) in the olfactory nerve layer, glomerular layer, and external plexiform layer. During long exposures (≈10 min), the initially dominant dorsal SAPs declined in intensity and area, whereas in some OB regions, the initially weak ventral/lateral SAPs increased first and then decreased. The SAPs of different concentrations were topologically similar, which implies that whereas an odor at various concentrations activates the same subsets of receptor cells, different concentrations are assessed and discriminated by variable magnitudes of laminarspecific activations. IAA and limonene reproducibly activated different subsets of receptor cells with some overlaps. Whereas qualitative topographical agreement was observed with results from other methods, the current dynamic blood oxygenation level-dependent functional MRI results can provide quantitative SAPs of the entire OB.
The principle of delivery of T cell epitopes to antigen-presenting cells applied to peptides from influenza virus, ovalbumin, and hen egg lysozyme: Implications for peptide vaccination
Authors: Rasmussen Ingunn B. , Lunde Elin , Michaelsen Terje E. , Bogen Bjarne , Sandlie Inger
Published: July 2002

Targeting of antigens to antigen-presenting cells (APCs) increases CD4+T cell activation, and this observation can be exploited in the development of new vaccines. We have chosen an antigen-targeting approach in which we make recombinant antibodies (Abs) with T cell epitopes in their constant region and APC-specific variable regions. Three commonly used model epitopes, amino acids 110–120 of hemagglutinin, 323–339 of ovalbumin, and 46–61 of hen egg lysozyme, were introduced as loops in the CH1 domain of human IgG3. For all three epitopes, we show that the recombinant molecules are secreted from transfected cells. The epitopes are presented to specific T cells, and targeting to IgD on B cellsin vitroenhances the presentation efficiency by 104to 105compared with the free peptide. After i.v. injection, the epitopes targeted to IgD are presented by splenic APCs to activate specific T cells, whereas little or no activation could be detected without targeting, even after the amount of antigen injected was increased 100-fold or more. Because a wide variety of T cell epitopes, in terms of both length and secondary structure, can be tolerated in loops in constant domains of Abs, the Ab constant region seems to have the intrinsic stability that is needed for this fusion molecule strategy. It might thus be possible to load the Ab with several different epitopes in loops in different domains and thereby make a targeted multisubunit vaccine.
Zinc induces a Src family kinase-mediated up-regulation of NMDA receptor activity and excitotoxicity
Authors: Manzerra Pat , Behrens M. Margarita , Canzoniero Lorella M. T. , Wang Xue Qing , Heidinger Valérie , Ichinose Tomomi , Yu Shan Ping , Choi Dennis W.
Published: July 2002

Zinc is coreleased with glutamate from excitatory nerve terminals
throughout the central nervous system and acutely inhibits
N
-methyl-
d
-aspartate (NMDA) receptor
activation. Here we report that cultured murine cortical neurons
briefly exposed to sublethal concentrations of zinc developed increased
intracellular free Na
+
, phosphorylation of Src kinase at
tyrosine 220, and tyrosine phosphorylation of NMDA receptor 2A/2B
subunits, in a fashion sensitive to the Src family kinase inhibitor
4-amino-5-(4-chlorophenyl)-7-(
t
-butyl)pyrazolo[3,4-d]pyrimidine,
PP2. Functionally, this zinc exposure produced a delayed increase in
NMDA receptor current in perforated patch but not conventional
whole-cell recordings, as well as an increase in NMDA receptor-mediated
cell death. These observations suggest that the effect of synaptically
released zinc on neuronal NMDA receptors may be biphasic: acute block,
followed by Src family kinase-mediated up-regulation of NMDA receptor
activity and cytotoxicity.
Vitamin A deprivation results in reversible loss of hippocampal long-term synaptic plasticity
Authors: Misner D. L. , Jacobs S. , Shimizu Y. , de Urquiza A. M. , Solomin L. , Perlmann T. , De Luca L. M. , Stevens C. F. , Evans R. M.
Published: July 2002

Despite its long history, the central effects of progressive
depletion of vitamin A in adult mice has not been previously described.
An examination of vitamin-deprived animals revealed a progressive and
ultimately profound impairment of hippocampal CA1 long-term
potentiation and a virtual abolishment of long-term depression.
Importantly, these losses are fully reversible by dietary vitamin A
replenishment
in vivo
or direct application of all
trans
-retinoic acid to acute hippocampal slices. We find
retinoid responsive transgenes to be highly active in the hippocampus,
and by using dissected explants, we show the hippocampus to be a site
of robust synthesis of bioactive retinoids. In aggregate, these results
demonstrate that vitamin A and its active derivatives function as
essential competence factors for long-term synaptic plasticity within
the adult brain, and suggest that key genes required for long-term
potentiation and long-term depression are retinoid dependent. These
data suggest a major mental consequence for the hundreds of millions of
adults and children who are vitamin A deficient.
Stone tool function at the paleolithic sites of Starosele and Buran Kaya III, Crimea: Behavioral implications
Authors: Hardy Bruce L. , Kay Marvin , Marks Anthony E. , Monigal Katherine
Published: July 2002

Stone tools are often the most abundant type of cultural remains at Paleolithic sites, yet their function is often poorly understood. Investigations of stone tool function, including microscopic use-wear and residue analyses, were performed on a sample of artifacts from the Paleolithic sites of Starosele (40,000–80,000 years BP) and Buran Kaya III (32,000–37,000 years BP). The Middle Paleolithic levels at Starosele exhibit a typical variant of the local Micoquian Industry. The artifacts from Buran Kaya III most closely resemble an Early Streletskayan Industry associated with the early Upper Paleolithic. The results of the functional analyses suggest that hominids at both sites were exploiting woody and starchy plant material as well as birds and mammals. Both sites show evidence of hafting of a wide variety of tools and the possible use of projectile or thrusting spears. These analyses were performed by using two different techniques conducted by independent researchers. Combined residue and use-wear analyses suggest that both the Upper Paleolithic and Middle Paleolithic hominids at these sites were broad-based foragers capable of exploiting a wide range of resources.
Measuring the thickness of the human cerebral cortex from magnetic resonance images
Authors: Fischl Bruce , Dale Anders M.
Published: July 2002

Accurate and automated methods for measuring the thickness of human
cerebral cortex could provide powerful tools for diagnosing and
studying a variety of neurodegenerative and psychiatric disorders.
Manual methods for estimating cortical thickness from neuroimaging data
are labor intensive, requiring several days of effort by a trained
anatomist. Furthermore, the highly folded nature of the cortex is
problematic for manual techniques, frequently resulting in measurement
errors in regions in which the cortical surface is not perpendicular to
any of the cardinal axes. As a consequence, it has been impractical to
obtain accurate thickness estimates for the entire cortex in individual
subjects, or group statistics for patient or control populations. Here,
we present an automated method for accurately measuring the thickness
of the cerebral cortex across the entire brain and for generating
cross-subject statistics in a coordinate system based on cortical
anatomy. The intersubject standard deviation of the thickness measures
is shown to be less than 0.5 mm, implying the ability to detect focal
atrophy in small populations or even individual subjects. The
reliability and accuracy of this new method are assessed by
within-subject test–retest studies, as well as by comparison of
cross-subject regional thickness measures with published values.
Classes of small-world networks
Authors: Amaral L. A. N. , Scala A. , Barthélémy M. , Stanley H. E.
Published: July 2002

We study the statistical properties of a variety of diverse
real-world networks. We present evidence of the occurrence of three
classes of small-world networks: (
a
) scale-free
networks, characterized by a vertex connectivity distribution that
decays as a power law; (
b
) broad-scale networks,
characterized by a connectivity distribution that has a power law
regime followed by a sharp cutoff; and (
c
) single-scale
networks, characterized by a connectivity distribution with a fast
decaying tail. Moreover, we note for the classes of broad-scale and
single-scale networks that there are constraints limiting the addition
of new links. Our results suggest that the nature of such constraints
may be the controlling factor for the emergence of different classes of
networks.
Identification of sites for exponential translation in living dendrites
Authors: Job Christy , Eberwine James
Published: July 2002

Neuronal processes contain mRNAs and membrane structures, and some forms of synaptic plasticity seem to require protein synthesis in dendrites of hippocampal neurons. To quantitate dendritic protein synthesis, we used multiphoton microscopy of green fluorescent protein synthesized in living isolated dendrites. Transfection of dendrites with mRNA encoding green fluorescent protein resulted in fluorescence that exponentially increased on stimulation with a glutamate receptor agonist; a reaction attenuated by the translation inhibitors anisomycin and emetine. Comparable experiments on whole neurons revealed that (RS)-3,5-dihydroxy-phenylglycine 0.5 H
2
O (DHPG)-stimulated fluorescence was linear in cell bodies relative to the exponential increase seen in dendrites. Detailed spatial analysis of the subdendritic distribution of fluorescence revealed “hotspots,” sites of dendritic translation that were spatially stable. However, detailed temporal analysis of these hotspots revealed heterogeneous rates of translation. A double-label protocol counterstaining for ribosomes indicated that sites of “fastest” translation correlated with increased ribosome density, consistent with ribosome subunit assembly for initiation, the first step of translation. We propose that dendrites have specific sites specialized for fast translation.
Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function
Authors: Zhou Qing , Clarke Lane , Nie Rong , Carnes Kay , Lai Li-Wen , Lien Yeong-Hau H. , Verkman Alan , Lubahn Dennis , Fisher Jane S. , Katzenellenbogen Benita S. , Hess Rex A.
Published: July 2002

Estrogen receptor α (ERα) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogen's bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogen's role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na+/H+exchanger-3 (NHE3) and the rate of22Na+transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased inERαknockout (αERKO) efferent ductules. Targeted gene-deficient mice were compared with αERKO, and theNHE3knockout andCAII-deficient mice showed αERKO-like fluid accumulation, but only theNHE3knockout and αERKO mice were infertile. Northern blot analysis showed decreases in mRNA forNHE3in αERKO and antiestrogen-treated mice. The changes in AQP1 and CAII in αERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in αERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na+transport. Finally, these data raise the possibility of targeting ERα in developing a contraceptive for the male.
Alzheimer's β-secretase, β-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase
Authors: Kitazume Shinobu , Tachida Yuriko , Oka Ritsuko , Shirotani Keiro , Saido Takaomi C. , Hashimoto Yasuhiro
Published: July 2002

The deposition of amyloid β-peptide (Aβ) in the brain is closely associated with the development of Alzheimer's disease. Aβ is generated from the amyloid precursor protein (APP) by sequential action of β-secretase (BACE1) and γ-secretase. Although BACE1 is distributed among various other tissues, its physiological substrates other than APP have yet to be identified. ST6Gal I is a sialyltransferase that produces a sialylα2,6galactose residue, and the enzyme is secreted out of the cell after proteolytic cleavage. We report here that BACE1 is involved in the proteolytic cleavage of ST6Gal I, on the basis of the following observations. ST6Gal I was colocalized with BACE1 in the Golgi apparatus by immunofluorescence microscopy, suggesting that BACE1 acts on ST6Gal I within the same intracellular compartment. When BACE1 was overexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I markedly increased. When APP
SW
(Swedish familial Alzheimer's disease mutation), a preferable substrate for BACE1, was coexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I significantly decreased, suggesting that that the β-cleavage of overexpressed APP
SW
competes with ST6Gal I processing. In addition, BACE1-Fc (Fc, the hinge and constant region of IgG) chimera cleaved protein A-ST6Gal I fusion protein
in vitro
. Thus, we conclude that BACE1 is responsible for the cleavage and secretion of ST6Gal I.
Acyl-CoA hydrolysis by the high molecular weight protein 1 subunit of yersiniabactin synthetase: Mutational evidence for a cascade of four acyl-enzyme intermediates during hydrolytic editing
Authors: Suo Zucai , Chen Huawei , Walsh Christopher T.
Published: July 2002

Yersiniabactin (Ybt) synthetase is a three-subunit, 17-domain
[7 domains in high molecular weight protein (HMWP)2, 9 in HMWP1, and 1
in YbtE] enzyme producing the virulence-conferring siderophore
yersiniabactin in
Yersinia pestis
. The 350-kDa HMWP1
subunit contains a polyketide synthase module
(KS-AT-MT
2
-KR-ACP) and a nonribosomal peptide synthetase
module (Cy
3
-MT
3
-PCP
3
-TE). The
full-length HMWP1 was heterologously overexpressed in
Escherichia coli
and purified to near homogeneity. The
purified HMWP1 showed thioesterase activity toward acyl-CoAs, such as
acetyl-CoA, benzoyl-CoA, and malonyl-CoA, with saturation kinetics and
relative catalytic efficiencies of 172:50:1. A chain-releasing
thioesterase (TE) activity is ascribed to the C-terminal TE domain, and
this was substantiated by the fact that
acyl-
N
-acetylcysteamines were hydrolyzed by the didomain
PCP
3
-TE fragment of HMWP1. However, PCP
3
-TE
failed to hydrolyze any of the acyl-CoAs, suggesting the TE domain does
not recognize CoA moiety, thus the acyl-CoA hydrolysis by HMWP1 must
involve other domains. Ser-to-Ala mutants in each of the AT, ACP,
PCP
3
, and TE domains reduced hydrolysis rates of the two
fastest substrates, acetyl-CoA and benzoyl-CoA, by more than two orders
of magnitude. Thus, the acyl-CoA hydrolysis activity requires 4 of the
9 domains of HMWP1, and it is consistent with autoacylation of the AT
domain active site serine and subsequent passage of the itinerant acyl
chain from AT to ACP to PCP
3
to the TE domain, a cascade of
four sequential acyl-enzyme intermediates, for hydrolytic turnover.
This could represent an editing pathway for this polyketide
synthase/nonribosomal peptide synthetase assembly line.
Cloning and functional expression of cDNAs encoding human and rat pancreatic polypeptide receptors.
Authors: Yan H , Yang J , Marasco J , Yamaguchi K , Brenner S , Collins F , Karbon W
Published: July 2002

PCR was used to isolate nucleotide sequences that may encode novel members of the neuropeptide Y receptor family. By use of a PCR product as a hybridization probe, a full-length human cDNA was isolated that encodes a 375-aa protein with a predicted membrane topology identifying it as a member of the G-protein-coupled receptor superfamily. After stable transfection of the cDNA into human embryonic kidney 293 cells, the receptor exhibited high affinity (Kd = 2.8 nM) for 125I-labeled human pancreatic polypeptide (PP). Competition binding studies in whole cells indicated the following rank order of potency: human PP = bovine PP > or = human [Pro34]peptide YY > rat PP > human peptide YY = human neuropeptide Y. Northern blot analysis revealed that human PP receptor mRNA is most abundantly expressed in skeletal muscle and, to a lesser extent, in lung and brain tissue. A rat cDNA clone encoding a high-affinity PP receptor that is 74% identical to the human PP receptor at the amino acid level was also isolated. These receptor clones will be useful in elucidating the functional role of PP and designing selective PP receptor agonists and antagonists.
Complementation of the beige mutation in cultured cells by episomally replicating murine yeast artificial chromosomes.
Authors: Perou C M , Justice M J , Pryor R J , Kaplan J
Published: July 2002

Chédiak-Higashi syndrome in man and the beige mutation of mice are phenotypically similar disorders that have profound effects upon lysosome and melanosome morphology and function. We isolated two murine yeast artificial chromosomes (YACs) that, when introduced into beige mouse fibroblasts, complement the beige mutation. The complementing YACs exist as extrachromosomal elements that are amplified in high concentrations of G418. When YAC-complemented beige cells were fused to human Chédiak-Higashi syndrome or Aleutian mink fibroblasts, complementation of the mutant phenotype also occurred. These results localize the beige gene to a 500-kb interval and demonstrate that the same or homologous genes are defective in mice, minks, and humans.
Assembly and movement of a plant virus carrying a green fluorescent protein overcoat.
Authors: Cruz S S , Chapman S , Roberts A G , Roberts I M , Prior D A , Oparka K J
Published: July 2002

Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread.