bluuebunny/crossref_metadata_2025 huggingface dataset:
Amphetamine-induced loss of human dopamine transporter activity: An internalization-dependent and cocaine-sensitive mechanism
Authors: Saunders Christine , Ferrer Jasmine V. , Shi Lei , Chen Jiayun , Merrill Gerald , Lamb Maria E. , Leeb-Lundberg L. M. Fredrik , Carvelli Lucia , Javitch Jonathan A. , Galli Aurelio
Published: July 2002

The dopamine transporter (DAT) is a target of amphetamine (AMPH) and cocaine. These psychostimulants attenuate DAT clearance efficiency, thereby increasing synaptic dopamine (DA) levels. Re-uptake rate is determined by the number of functional transporters at the cell surface as well as by their turnover rate. Here, we present evidence that DAT substrates, including AMPH and DA, cause internalization of human DAT, thereby reducing transport capacity. Acute treatment with AMPH reduced the maximal rate of [
3
H]DA uptake, decreased AMPH-induced currents, and significantly redistributed the immunofluorescence of an epitope-tagged DAT from the plasma membrane to the cytosol in human embryonic kidney 293 cells. Conversely, DAT inhibitors, such as cocaine, mazindol, and nomifensine, when administered with AMPH, blocked the reduction in [
3
H]DA uptake and the redistribution of DAT immunofluorescence to the cytosol. The reductions of [
3
H]DA uptake and AMPH-induced DAT internalization also were inhibited by coexpression of a dominant negative mutant of dynamin I (K44A), indicating that endocytosis modulates transport capacity, likely through a clathrin-mediated pathway. With this mechanism of regulation, acute application of AMPH would reduce DA uptake not only by direct competition for uptake, but also by reducing the available cell-surface DAT. Moreover, AMPH-induced internalization might diminish the amount of DAT available for DA efflux, thereby modulating the cytotoxic effects of elevated extracellular DA.
Shear stress-induced apoptosis of adherent neutrophils: A mechanism for persistence of cardiovascular device infections
Authors: Shive Matthew S. , Salloum Mariah L. , Anderson James M.
Published: July 2002

The mechanisms underlying problematic cardiovascular device-associated infections are not understood. Because the outcome of the acute response to infection is largely dependent on the function of neutrophils, the persistence of these infections suggests that neutrophil function may be compromised because of cellular responses to shear stress. A rotating disk system was used to generate physiologically relevant shear stress levels (0–18 dynes/cm2; 1 dyne = 10 μN) at the surface of a polyetherurethane urea film. We demonstrate that shear stress diminishes phagocytic ability in neutrophils adherent to a cardiovascular device material, and causes morphological and biochemical alterations that are consistent with those described for apoptosis. Complete neutrophil apoptosis occurred at shear stress levels above 6 dynes/cm2after only 1 h. Morphologically, these cells displayed irreversible cytoplasmic and nuclear condensation while maintaining intact membranes. Analysis of neutrophil area and filamentous actin content demonstrated concomitant decreases in both cell area and actin content with increasing levels of shear stress. Neutrophil phagocytosis of adherent bacteria decreased with increasing shear stress. Biochemical alterations included membrane phosphatidylserine exposure and DNA fragmentation, as evaluated byin situannexin V and terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) assays, respectively. The potency of the shear-stress effect was emphasized by comparative inductive studies with adherent neutrophils under static conditions. The combination of tumor necrosis factor-α and cycloheximide was ineffective in inducing >21% apoptosis after 3 h. These findings suggest a mechanism through which shear stress plays an important role in the development of bacterial infections at the sites of cardiovascular device implantation.
Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins
Authors: Ong S. H. , Hadari Y. R. , Gotoh N. , Guy G. R. , Schlessinger J. , Lax I.
Published: July 2002

The docking protein FRS2 is a major downstream effector that links
fibroblast growth factor (FGF) and nerve growth factor receptors with
the Ras/mitogen-activated protein kinase signaling cascade. In this
report, we demonstrate that FRS2 also plays a pivotal role in
FGF-induced recruitment and activation of phosphatidylinositol 3-kinase
(PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2α
leads to Grb2-mediated complex formation with the docking protein Gab1
and its tyrosine phosphorylation, resulting in the recruitment and
activation of PI3-kinase. Furthermore, Grb2 bound to
tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily
via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1
and via its amino-terminal SH3 domain with the nucleotide exchange
factor Sos1. Assembly of FRS2α:Grb2:Gab1 complex induced by FGF
stimulation results in activation of PI3-kinase and downstream effector
proteins such as the S/T kinase Akt, whose cellular localization and
activity are regulated by products of PI3-kinase. These experiments
reveal a unique mechanism for generation of signal diversity by growth
factor-induced coordinated assembly of a multidocking protein complex
that can activate the Ras/mitogen-activated protein kinase cascade to
induce cell proliferation and differentiation, and PI3-kinase to
activate a mediator of a cell survival pathway.
Platelets modulate gastric ulcer healing: Role of endostatin and vascular endothelial growth factor release
Authors: Ma Li , Elliott Susan N. , Cirino Giuseppe , Buret Andre , Ignarro Louis J. , Wallace John L.
Published: July 2002

Bleeding and delayed healing of ulcers are well recognized clinical
problems associated with the use of aspirin and other nonsteroidal
antiinflammatory drugs, which have been attributed to their
antiaggregatory effects on platelets. We hypothesized that antiplatelet
drugs might interfere with gastric ulcer healing by suppressing the
release of growth factors, such as vascular endothelial growth factor
(VEGF), from platelets. Gastric ulcers were induced in rats by serosal
application of acetic acid. Daily oral treatment with vehicle, aspirin,
or ticlopidine (an ADP receptor antagonist) was started 3 days later
and continued for 1 week. Ulcer induction resulted in a significant
increase in serum levels of VEGF and a significant decrease in serum
levels of endostatin (an antiangiogenic factor). Although both aspirin
and ticlopidine markedly suppressed platelet aggregation, only
ticlopidine impaired gastric ulcer healing and angiogenesis as well as
reversing the ulcer-associated changes in serum levels of VEGF and
endostatin. The effects of ticlopidine on ulcer healing and
angiogenesis were mimicked by immunodepletion of circulating platelets,
and ticlopidine did not influence ulcer healing when given to
thrombocytopenic rats. Incubation of human umbilical vein endothelial
cells with serum from ticlopidine-treated rats significantly reduced
proliferation and increased apoptosis, effects reversed by an
antibody directed against endostatin. Ticlopidine treatment resulted in
increased platelet endostatin content and release. These results
demonstrate a previously unrecognized contribution of platelets to the
regulation of gastric ulcer healing. Such effects likely are mediated
through the release from platelets of endostatin and possibly VEGF. As
shown with ticlopidine, drugs that influence gastric ulcer healing may
do so in part through altering the ability of platelets to release
growth factors.
Folding and signaling share the same pathway in a photoreceptor
Authors: Lee Byoung-Chul , Pandit Adarsh , Croonquist Paula A. , Hoff Wouter D.
Published: July 2002

The photoreceptor photoactive yellow protein (PYP) was used as a model system to study receptor activation and protein folding. Refolding was studied by stopped-flow absorbance spectroscopy for PYP with either a trans or a cis chromophore. Chromophore trans to cis isomerization, the mechanism of light detection by PYP, greatly affects the protein folding process. When the cis chromophore is present, refolding from the unfolded state proceeds through the putative signaling state of PYP as an on-pathway intermediate. In addition, moderate denaturant concentrations result in the specific unfolding of the signaling state of PYP. Thus, the signaling state is common to the pathways of folding and signaling. This result provides an avenue for the study of protein folding. We demonstrate how this approach can be used to establish whether a folding intermediate is on-pathway or off-pathway. The results also reveal transient partial unfolding as a molecular mechanism for signaling.
Quantification of the hydrophobic interaction by simulations of the aggregation of small hydrophobic solutes in water
Authors: Raschke Tanya M. , Tsai Jerry , Levitt Michael
Published: July 2002

The hydrophobic interaction, the tendency for nonpolar molecules to
aggregate in solution, is a major driving force in biology. In a direct
approach to the physical basis of the hydrophobic effect, nanosecond
molecular dynamics simulations were performed on increasing numbers of
hydrocarbon solute molecules in water-filled boxes of different sizes.
The intermittent formation of solute clusters gives a free energy that
is proportional to the loss in exposed molecular surface area with a
constant of proportionality of 45 ± 6
cal/mol⋅Å
2
. The molecular surface area is the
envelope of the solute cluster that is impenetrable by solvent and is
somewhat smaller than the more traditional solvent-accessible surface
area, which is the area transcribed by the radius of a solvent molecule
rolled over the surface of the cluster. When we apply a factor relating
molecular surface area to solvent-accessible surface area, we obtain 24
cal/mol⋅Å
2
. Ours is the first direct calculation,
to our knowledge, of the hydrophobic interaction from molecular
dynamics simulations; the excellent qualitative and quantitative
agreement with experiment proves that simple van der Waals interactions
and atomic point-charge electrostatics account for the most important
driving force in biology.
Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative β isoform: A mechanism for the generation of glucocorticoid resistance
Authors: Webster Jeffrey C. , Oakley Robert H. , Jewell Christine M. , Cidlowski John A.
Published: July 2002

Inflammatory responses in many cell types are coordinately
regulated by the opposing actions of NF-κB and the glucocorticoid
receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two
protein isoforms: a cytoplasmic alpha form (GRα), which binds
hormone, translocates to the nucleus, and regulates gene transcription,
and a nuclear localized beta isoform (GRβ), which does not bind known
ligands and attenuates GRα action. We report here the identification
of a tumor necrosis factor (TNF)-responsive NF-κB DNA binding site 5′
to the hGR promoter that leads to a 1.5-fold increase in GRα mRNA and
a 2.0-fold increase in GRβ mRNA in HeLaS3 cells, which
endogenously express both GR isoforms. However, TNF-α
treatment disproportionately increased the steady-state levels of the
GRβ protein isoform over GRα, making GRβ the predominant
endogenous receptor isoform. Similar results were observed
following treatment of human CEMC7 lymphoid cells with TNF-α or IL-1.
The increase in GRβ protein expression correlated with the
development of glucocorticoid resistance.
Regulation of alternative splicing by a transcriptional enhancer through RNA pol II elongation
Authors: Kadener Sebastián , Fededa Juan Pablo , Rosbash Michael , Kornblihtt Alberto R.
Published: July 2002

Promoters and enhancers are cis-acting elements that control gene transcription via complex networks of protein–DNA and protein–protein interactions. Whereas promoters deal with putting in place the RNA polymerase, both enhancers and promoters can control transcriptional initiation and elongation. We have previously shown that promoter structure modulates alternative splicing, strengthening the concept of a physical and functional coupling between transcription and splicing. Here we report that the promoter effect is due to the control of RNA pol II elongation. We found that the simian virus 40 (SV40) transcriptional enhancer, inserted in fibronectin (FN) minigene constructs transfected into mammalian cells, controls alternative splicing by inhibiting inclusion of the FN extra domain I (EDI) exon into mature mRNA. Deletion analysis of enhancer subdomains and competitions
in vivo
with excess of specific enhancer DNA subfragments demonstrate that the “minimal” enhancer, consisting of two 72-bp repeats, is responsible for the splicing effect. The 72-bp repeat region has been reported to promote RNA pol II elongation. When transcription is driven by the α-globin promoter linked to the SV40 enhancer, basal EDI inclusion and activation by the SR (Ser–Arg-rich) protein SF2/ASF are much lower than with other promoters. Deletion of only one of the two 72-bp repeats not only provokes higher EDI inclusion levels but allows responsiveness to SF2/ASF. These effects are the consequence of a decrease in RNA pol II elongation evidenced both by an increase in the proportions of shorter proximal over full length transcripts and by higher pol II densities upstream of the alternative exon detected by chromatin immunoprecipitation.
Light regulation of type IV pilus-dependent motility by chemosensor-like elements in
Synechocystis
PCC6803
Authors: Bhaya Devaki , Takahashi Akiko , Grossman Arthur R.
Published: July 2002

To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium
Synechocystis
PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an “inverted motility response” (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis.
Synechocystis
PCC6803 has three loci with chemotaxis-like genes, of which two,
Tax1
and
Tax3
, are involved in phototaxis. Transposons interrupting the
Tax1
locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for
taxAY1
was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the
Tax3
locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.
A quantitative, high-throughput screen for protein stability
Authors: Ghaemmaghami S. , Fitzgerald M. C. , Oas T. G.
Published: July 2002

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric λ repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.
Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death
Authors: Suzuki Yasuyuki , Nakabayashi Yui , Takahashi Ryosuke
Published: July 2002

The inhibitor of apoptosis (IAP) family of anti-apoptotic proteins regulate programmed cell death and/or apoptosis. One such protein, X-linked IAP (XIAP), inhibits the activity of the cell death proteases, caspase-3, -7, and -9. In this study, using constitutively active mutants of caspase-3, we found that XIAP promotes the degradation of active-form caspase-3, but not procaspase-3, in living cells. The XIAP mutants, which cannot interact with caspase-3, had little or no activity of promoting the degradation of caspase-3. RING finger mutants of XIAP also could not promote the degradation of caspase-3. A proteasome inhibitor suppressed the degradation of caspase-3 by XIAP, suggesting the involvement of a ubiquitin-proteasome pathway in the degradation. An
in vitro
ubiquitination assay revealed that XIAP acts as a ubiquitin-protein ligase for caspase-3. Caspase-3 was ubiquitinated in the presence of XIAP in living cells. Both the association of XIAP with caspase-3 and the RING finger domain of XIAP were essential for ubiquitination. Finally, the RING finger mutants of XIAP were less effective than wild-type XIAP at preventing apoptosis induced by overexpression of either active-form caspase-3 or Fas. These results demonstrate that the ubiquitin-protein ligase activity of XIAP promotes the degradation of caspase-3, which enhances its anti-apoptotic effect.
Molecular basis of lutropin recognition by the mannose/GalNAc-4-SO4receptor
Authors: Roseman Daniel S. , Baenziger Jacques U.
Published: July 2002

The circulatory half-life of the glycoprotein hormone lutropin (LH) is precisely regulated by the mannose (Man)/GalNAc-4-SO4receptor expressed in hepatic endothelial cells. Rapid clearance from the circulation contributes to the episodic rise and fall of LH levels that is essential for maximal stimulation of the G protein-coupled LH receptor. We have defined two molecular forms of the Man/GalNAc-4-SO4receptor that differ in ligand specificity, cell and tissue expression, and function. The form expressed by hepatic endothelial cells binds GalNAc-4-SO4-bearing ligands and regulates hormone circulatory half-life, whereas the form expressed by macrophages binds Man-bearing ligands and may play a role in innate immunity. We demonstrate that the GalNAc-4-SO4-specific form in hepatic endothelial cells is dimeric whereas the Man-specific form in lung macrophages is monomeric, accounting for the different ligand specificities of the receptor expressed in these tissues. Two cysteine-rich domains, each of which binds a single GalNAc-4-SO4, are required to form stable complexes with LH. The kinetics of LH binding by the GalNAc-4-SO4-specific form of the receptor in conjunction with its rate of internalization from the cell surface make it likely that only two of the four terminal GalNAc-4-SO4moieties present on native LH are engaged before receptor internalization. As a result, the rate of hormone clearance will remain constant over a wide range of LH concentrations and will not be sensitive to variations in the number of terminal GalNAc-4-SO4moieties as long as two or more are present on multiple oligosaccharides.
Neural agrin controls acetylcholine receptor stability in skeletal muscle fibers
Authors: Bezakova Gabriela , Rabben Inger , Sefland Iren , Fumagalli Guido , Lømo Terje
Published: July 2002

At mammalian neuromuscular junctions (NMJs), innervation induces
and maintains the metabolic stability of acetylcholine receptors
(AChRs). To explore whether neural agrin may cause similar receptor
stabilization, we injected neural agrin cDNA of increasing transfection
efficiencies into denervated adult rat soleus (SOL) muscles. As the
efficiency increased, the amount of recombinant neural agrin expressed
in the muscles also increased. This agrin aggregated AChRs on muscle
fibers, whose half-life increased in a dose-dependent way from 1 to 10
days. Electrical muscle stimulation enhanced the stability of AChRs
with short half-lives. Therefore, neural agrin can stabilize aggregated
AChRs in a concentration- and activity-dependent way. However, there
was no effect of stimulation on AChRs with a long half-life (10 days).
Thus, at sufficiently high concentrations, neural agrin alone can
stabilize AChRs to levels characteristic of innervated NMJs.
A src family tyrosine kinase inhibits neurotransmitter release from neuronal cells
Authors: Ohnishi Hiroshi , Yamamori Saori , Ono Kanako , Aoyagi Kyota , Kondo Shunzo , Takahashi Masami
Published: July 2002

Tyrosine kinases are expressed in many tissues, particularly in the central nervous system, and regulate various cellular functions. We report here that a src family tyrosine kinase-specific inhibitor, PP2, enhances neurotransmitter release from PC12 cells and primary cultured neurons. PP2 enhances only Ca
2+
-dependent release; it does not affect basal release. These effects result from an enhancement of vesicular exocytosis and not from the reuptake or refilling of neurotransmitters because Ca
2+
-dependent secretion of an exogenously expressed reporter protein, the human growth hormone (hGH), is also enhanced by PP2. Overexpression of constitutive active v-src, but not of a kinase-inactive mutant, suppressed Ca
2+
-dependent release. In PP2-treated cells, Pyk2, paxillin, and some other proteins showed a decrease in tyrosine phosphorylation, and the enhancement of tyrosine phosphorylation of these proteins in response to Ca
2+
influx was also reduced. Electron and fluorescence microscopy showed that PP2 treatment induced morphological change and decreased phalloidin reactivity at the filopodium-like structures on the processes of PC12 cells. Interestingly, inhibition of actin polymerization with cytochalasin D and latrunculin A enhanced Ca
2+
-dependent, but not basal, release. It is possible that a src family tyrosine kinase, through the regulation of actin dynamics, has an inhibitory function to regulate neurotransmitter release.
X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs
Authors: Ha Ya , Stevens David J. , Skehel John J. , Wiley Don C.
Published: July 2002

The three-dimensional structures of avian H5 and swine H9 influenza
hemagglutinins (HAs) from viruses closely related to those that caused
outbreaks of human disease in Hong Kong in 1997 and 1999 were
determined bound to avian and human cell receptor analogs. Emerging
influenza pandemics have been accompanied by the evolution of
receptor-binding specificity from the preference of avian viruses for
sialic acid receptors in α2,3 linkage to the preference of human
viruses for α2,6 linkages. The four new structures show that HA
binding sites specific for human receptors appear to be wider than
those preferring avian receptors and how avian and human receptors are
distinguished by atomic contacts at the glycosidic linkage.
α2,3-Linked sialosides bind the avian HA in a trans conformation to
form an α2,3 linkage-specific motif, made by the glycosidic oxygen
and 4-OH of the penultimate galactose, that is complementary to the
hydrogen-bonding capacity of Gln-226, an avian-specific residue.
α2,6-Linked sialosides bind in a cis conformation, exposing the
glycosidic oxygen to solution and nonpolar atoms of the receptor to
Leu-226, a human-specific residue. The new structures are compared with
previously reported crystal structures of HA/sialoside complexes of
the H3 subtype that caused the 1968 Hong Kong Influenza virus pandemic
and analyzed in relation to HA sequences of all 15 subtypes and to
receptor affinity data to make clearer how receptor-binding sites of
HAs from avian viruses evolve as the virus adapts to humans.
The free energy landscape for β hairpin folding in explicit water
Authors: Zhou Ruhong , Berne Bruce J. , Germain Robert
Published: July 2002

The folding free energy landscape of the C-terminal β
hairpin of protein G has been explored in this study with explicit
solvent under periodic boundary condition and
oplsaa
force
field. A highly parallel replica exchange method that combines
molecular dynamics trajectories with a temperature exchange Monte Carlo
process is used for sampling with the help of a new efficient algorithm
P3ME/RESPA. The simulation results show that the hydrophobic core and
the β strand hydrogen bond form at roughly the same time. The free
energy landscape with respect to various reaction coordinates is found
to be rugged at low temperatures and becomes a smooth funnel-like
landscape at about 360 K. In contrast to some very recent studies, no
significant helical content has been found in our simulation at all
temperatures studied. The β hairpin population and hydrogen-bond
probability are in reasonable agreement with the experiment at
biological temperature, but both decay more slowly than the experiment
with temperature.
Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression
Authors: Kim Chong-Hyun , Chung Hee Jung , Lee Hey-Kyoung , Huganir Richard L.
Published: July 2002

The interaction of PDZ domain-containing proteins with the C
termini of α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)
receptors has been suggested to be important in the regulation of
receptor targeting to excitatory synapses. Recent studies have shown
that the rapid internalization of AMPA receptors at synapses may
mediate, at least in part, the expression of long-term depression
(LTD). We have previously shown that phosphorylation of Ser-880 on the
AMPA receptor GluR2 subunit differentially regulated the interaction of
GluR2 with the PDZ domain-containing proteins GRIP1 and PICK1. Here, we
show that induction of LTD in hippocampal slices increases
phosphorylation of Ser-880 within the GluR2 C-terminal PDZ ligand,
suggesting that the modulation of GluR2 interaction with GRIP1 and
PICK1 may regulate AMPA receptor internalization during LTD. Moreover,
postsynaptic intracellular perfusion of GluR2 C-terminal peptides that
disrupt GluR2 interaction with PICK1 inhibit the expression of
hippocampal LTD. These results suggest that the interaction of GluR2
with PICK1 may play a regulatory role in the expression of LTD in
the hippocampus.
Quantifying genetic and nongenetic contributions to malarial infection in a Sri Lankan population
Authors: Mackinnon Margaret J. , Gunawardena D. M. , Rajakaruna Jagath , Weerasingha Sudath , Mendis Kamini N. , Carter Richard
Published: July 2002

Explaining the causes of variation in the severity of malarial disease remains a major challenge in the treatment and control of malaria. Many factors are known to contribute to this variation, including parasite genetics, host genetics, acquired immunity, and exposure levels. However, the relative importance of each of these to the overall burden of malarial disease in human populations has not been assessed. Here, we have partitioned variation in the incidence of malarial infection and the clinical intensity of malarial disease in a rural population in Sri Lanka into its component causes by pedigree analysis of longitudinal data. We found that human genetics, housing, and predisposing systematic effects (e.g., sex, age, occupation, history of infections, village) each explained approximately 15% of the variation in the frequency of malarial infection. For clinical intensity of illness, 20% of the variation was explained by repeatable differences between patients, about half of which was attributable to host genetics. The other half was attributable to semipermanent differences among patients, most of which could be explained by known predisposing factors. Three percent of variation in clinical intensity was explained by housing, and an additional 7% was explained by current influences relating to infection status (e.g., parasitemia, parasite species). Genetic control ofPlasmodium falciparuminfections appeared to modulate the frequency and intensity of infections, whereas genetic control ofPlasmodium vivaxinfections appeared to confer absolute susceptibility or refractoriness but not intensity of disease. Overall, the data show consistent, repeatable differences among hosts in their susceptibility to clinical disease, about half of which are attributable to host genes.
Internal initiation in
Saccharomyces cerevisiae
mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element
Authors: Thompson Sunnie R. , Gulyas Keith D. , Sarnow Peter
Published: July 2002

Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules. Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast
Saccharomyces cerevisiae
. We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second
URA3
cistron in dicistronic mRNAs in
S. cerevisiae
, thereby conferring uracil-independent growth. Curiously, the IRES functions poorly in wild-type yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA
met
genes disrupted. Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP/initiator tRNA
met
complexes. Furthermore, tRNA
met
-independent initiation was also observed in translation-competent extracts prepared from
S. cerevisiae
in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes. Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in
S. cerevisiae
in the absence of eIF2 and initiator tRNA
met
, by the same mechanism of factor-independent ribosome recruitment used in mammalian cells. These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry.
Genetic shift in photoperiodic response correlated with global warming
Authors: Bradshaw William E. , Holzapfel Christina M.
Published: July 2002

To date, all altered patterns of seasonal interactions observed in
insects, birds, amphibians, and plants associated with global warming
during the latter half of the 20th century are explicable as variable
expressions of plastic phenotypes. Over the last 30 years, the
genetically controlled photoperiodic response of the pitcher-plant
mosquito,
Wyeomyia smithii
, has shifted toward shorter,
more southern daylengths as growing seasons have become longer. This
shift is detectable over a time interval as short as 5 years. Faster
evolutionary response has occurred in northern populations where
selection is stronger and genetic variation is greater than in southern
populations.
W. smithii
represents an example of
actual genetic differentiation of a seasonality trait that is
consistent with an adaptive evolutionary response to recent global
warming.
Taxol biosynthesis: Taxane 13α-hydroxylase is a cytochrome P450-dependent monooxygenase
Authors: Jennewein Stefan , Rithner Christopher D. , Williams Robert M. , Croteau Rodney B.
Published: July 2002

A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, usingTaxus(yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10β-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using theSpodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5α-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10β-hydroxylase) as a taxane 13α-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.
BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia
Authors: Plesnila Nikolaus , Zinkel Sandra , Le Dean A. , Amin-Hanjani Sepideh , Wu Yonqin , Qiu Jianhua , Chiarugi Alberto , Thomas Sunu S. , Kohane Daniel S. , Korsmeyer Stanley J. , Moskowitz Michael A.
Published: July 2002

Mitochondria and cytochrome
c
release play a
role in the death of neurons and glia after cerebral ischemia. In the
present study, we investigated whether BID, a proapoptotic
promoter of cytochrome
c
release and caspase 8
substrate, was expressed in brain, activated after an ischemic insult
in vivo
and
in vitro
, and contributed to
ischemic cell death. We detected BID in the cytosol of mouse brain and
primary cultured mouse neurons and demonstrated, by using recombinant
caspase 8, that neuronal BID also is a caspase 8 substrate. After
2 h of oxygen/glucose deprivation, BID cleavage was
detected in neurons concurrent with caspase 8 activation but before
caspase 3 cleavage.
Bid
−/−
neurons were
resistant to death after oxygen/glucose deprivation, and caspase 3
cleavage was significantly reduced; however, caspase 8 cleavage did not
differ from wild type.
In vivo
, BID was cleaved 4 h
after transient middle cerebral artery occlusion. Infarct volumes and
cytochrome
c
release also were less in
Bid
−/−
mice (−67% and −41%, respectively)
after mild focal ischemia. These findings suggest that BID and
the mitochondrial-amplification pathway promoting caspase activation
contributes importantly to neuronal cell death after ischemic insult.
Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver.
Authors: Guidotti L G , Borrow P , Hobbs M V , Matzke B , Gresser I , Oldstone M B , Chisari F V
Published: July 2002

Hepatitis B virus (HBV) infection is thought to be controlled by virus-specific cytotoxic T lymphocytes (CTL). We have recently shown that HBV-specific CTL can abolish HBV replication noncytopathically in the liver of transgenic mice by secreting tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) after antigen recognition. We now demonstrate that hepatocellular HBV replication is also abolished noncytopathically during lymphocytic choriomeningitis virus (LCMV) infection, and we show that this process is mediated by TNF-alpha and IFN-alpha/beta produced by LCMV-infected hepatic macrophages. These results confirm the ability of these inflammatory cytokines to abolish HBV replication; they elucidate the mechanism likely to be responsible for clearance of HBV in chronically infected patients who become superinfected by other hepatotropic viruses; they suggest that pharmacological activation of intrahepatic macrophages may have therapeutic value in chronic HBV infection; and they raise the possibility that conceptually similar events may be operative in other viral infections as well.
A synthetic random basic copolymer with promiscuous binding to class II major histocompatibility complex molecules inhibits T-cell proliferative responses to major and minor histocompatibility antigens in vitro and confers the capacity to prevent murine graft-versus-host disease in vivo.
Authors: Schlegel P G , Aharoni R , Chen Y , Chen J , Teitelbaum D , Arnon R , Sela M , Chao N J
Published: July 2002

Graft-versus-host disease (GVHD) is a T-cell-mediated disease of transplanted donor T cells recognizing host alloantigens. Data presented in this report show, to our knowledge, for the first time that a synthetic copolymer of the amino acids L-Glu, L-Lys, L-Ala, and L-Tyr (molecular ratio, 1.9:6.0:4.7:1.0; Mr, 6000-8500) [corrected], termed GLAT, with promiscuous binding to multiple major histocompatibility complex class II alleles is capable of preventing lethal GVHD in the B10.D2 --> BALB/c model (both H-2d) across minor histocompatibility barriers. Administration of GLAT over a limited time after transplant significantly reduced the incidence, onset, and severity of disease. GLAT also improved long-term survival from lethal GVHD: 14/25 (56%) of experimental mice survived > 140 days after transplant compared to 2/26 of saline-treated or to 1/10 of hen egg lysozyme-treated control mice (P < 0.01). Long-term survivors were documented to be fully chimeric by PCR analysis of a polymorphic microsatellite region in the interleukin 1beta gene. In vitro, GLAT inhibited the mixed lymphocyte culture in a dose-dependent fashion across a variety of major barriers tested. Furthermore, GLAT inhibited the response of nylon wool-enriched T cells to syngeneic antigen-presenting cells presenting minor histocompatibility antigens. Prepulsing of the antigen-presenting cells with GLAT reduced the proliferative response, suggesting that GLAT inhibits antigen presentation.
Growth retardation and cysteine deficiency in gamma-glutamyl transpeptidase-deficient mice.
Authors: Lieberman M W , Wiseman A L , Shi Z Z , Carter B Z , Barrios R , Ou C N , Chévez-Barrios P , Wang Y , Habib G M , Goodman J C , Huang S L , Lebovitz R M , Matzuk M M
Published: July 2002

gamma-Glutamyl transpeptidase (GGT) is an ectoenzyme that catalyzes the first step in the cleavage of glutathione (GSH) and plays an essential role in the metabolism of GSH and GSH conjugates of carcinogens, toxins, and eicosanoids. To learn more about the role of GGT in metabolism in vivo, we used embryonic stem cell technology to generate GGT-deficient (GGTm1/GGTm1) mice. GGT-deficient mice appear normal at birth but grow slowly and by 6 weeks are about half the weight of wild-type mice. They are sexually immature, develop cataracts, and have coats with a gray cast. Most die between 10 and 18 weeks. Plasma and urine GSH levels in the GGTm1/GGTm1 mice are elevated 6-fold and 2500-fold, respectively, compared with wild-type mice. Tissue GSH levels are markedly reduced in eye, liver, and pancreas. Plasma cyst(e)ine levels in GGTm1/GGTm1 mice are reduced to approximately 20% of wild-type mice. Oral administration of N-acetylcysteine to GGTm1/GGTm1 mice results in normal growth rates and partially restores the normal agouti coat color. These findings demonstrate the importance of GGT and the gamma-glutamyl cycle in cysteine and GSH homeostasis.
Norepinephrine transporters have channel modes of conduction.
Authors: Galli A , Blakely R D , DeFelice L J
Published: July 2002

Neurotransmitter transporters couple to existing ion gradients to achieve reuptake of transmitter into presynaptic terminals. For coupled cotransport, substrates and ions cross the membrane in fixed stoichiometry. This is in contrast to ion channels, which carry an arbitrary number of ions depending on the channel open time. Members of the gamma-aminobutyric acid transporter gene family presumably function with fixed stoichiometry in which a set number of ions cotransport with one transmitter molecule. Here we report channel-like events from a presumably fixed stoichiometry [norepinephrine (NE)+, Na+, and Cl-], human NE (hNET) in the gamma-aminobutyric acid transporter gene family. These events are stimulated by NE and by guanethidine, an hNET substrate, and they are blocked by cocaine and the antidepressant desipramine. Voltage-clamp data combined with NE uptake data from these same cells indicate that hNETs have two functional modes of conduction: a classical transporter mode (T-mode) and a novel channel mode (C-mode). Both T-mode and C-mode are gated by the same substrates and antagonized by the same blockers. T-mode is putatively electrogenic because the transmitter and cotransported ions sum to one net charge. However, C-mode carries virtually all of the transmitter-induced current, even though it occurs with low probability. This is because each C-mode opening transports hundreds of charges per event. The existence of a channel mode of conduction in a previously established fixed-stoichiometry transporter suggests the appearance of an aqueous pore through the transporter protein during the transport cycle and may have significance for transporter regulation.
Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.
Authors: Schaefer A L , Val D L , Hanzelka B L , Cronan J E , Greenberg E P
Published: July 2002

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.
Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM.
Authors: Vogt A B , Kropshofer H , Moldenhauer G , Hämmerling G J
Published: July 2002

The nonclassical major histocompatibility complex class II molecule HLA-DM (DM) has recently been shown to play a central role in the class II-associated antigen presentation pathway: DM releases invariant chain-derived CLIP peptides (class II-associated invariant chain protein peptide) from HLA-DR (DR) molecules and thereby facilitates loading with antigenic peptides. Some observations have led to the suggestion that DM acts in a catalytic manner, but so far direct proof is missing. Here, we investigated in vitro the kinetics of exchange of endogenously bound CLIP for various peptides on DR1 and DR2a molecules: we found that in the presence of DM the peptide loading process follows Michaelis-Menten kinetics with turnover numbers of 3-12 DR molecules per minute per DM molecule, and with KM values of 500-1000 nM. In addition, surface plasmon resonance measurements showed that DM interacts efficiently with DR-CLIP complexes but only weakly with DR-peptide complexes isolated from DM-positive cells. Taken together, our data provide evidence that DM functions as an enzyme-like catalyst of peptide exchange and favors the generation of long-lived DR-peptide complexes that are no longer substrates for DM.
Preserved neuron number in the hippocampus of aged rats with spatial learning deficits.
Authors: Rapp P R , Gallagher M
Published: July 2002

Hippocampal neuron loss is widely viewed as a hallmark of normal aging. Moreover, neuronal degeneration is thought to contribute directly to age-related deficits in learning and memory supported by the hippocampus. By taking advantage of improved methods for quantifying neuron number, the present study reports evidence challenging these long-standing concepts. The status of hippocampal-dependent spatial learning was evaluated in young and aged Long-Evans rats using the Morris water maze, and the total number of neurons in the principal cell layers of the dentate gyrus and hippocampus was quantified according to the optical fractionator technique. For each of the hippocampal fields, neuron number was preserved in the aged subjects as a group and in aged individuals with documented learning and memory deficits indicative of hippocampal dysfunction. The findings demonstrate that hippocampal neuronal degeneration is not an inevitable consequence of normal aging and that a loss of principal neurons in the hippocampus fails to account for age-related learning and memory impairment. The observed preservation of neuron number represents an essential foundation for identifying the neurobiological effects of hippocampal aging that account for cognitive decline.
Protective immunity to HIV-1 in SCID/beige mice reconstituted with peripheral blood lymphocytes of exposed but uninfected individuals
Authors: Zhang Chengsheng , Cui Yan , Houston Stan , Chang Lung-Ji
Published: July 2002

Immunodeficiency typically appears many years after initial HIV infection. This long, essentially asymptomatic period contributes to the transmission of HIV in human populations. In rare instances, clearance of HIV-1 infection has been observed, particularly in infants. There are also reports of individuals who have been frequently exposed to HIV-1 but remain seronegative for the virus, and it has been hypothesized that these individuals are resistant to infection by HIV-1. However, little is known about the mechanism of immune clearance or protection against HIV-1 in these high-risk individuals because it is difficult to directly demonstratein vivoprotective immunity. Although most of these high-risk individuals show an HIV-1-specific cell-mediated immune response usingin vitroassays, their peripheral blood lymphocytes (PBLs) are still susceptible to HIV infection in tissue culture. To study this furtherin vivo, we have established a humanized SCID mouse infection model whereby T-, B-, and natural killer-cell defective SCID/beige mice that have been reconstituted with normal human PBLs can be infected with HIV-1. When the SCID/beige mice were reconstituted with PBLs from two different multiply exposed HIV-1 seronegative individuals, the mice showed resistance to infection by two strains of HIV-1 (macrophage tropic and T cell tropic), although the same PBLs were easily infectedin vitro. Mice reconstituted with PBLs from non-HIV-exposed controls were readily infected. When the same reconstituted mice were depleted of human CD8 T cells, however, they became susceptible to HIV-1 infection, indicating that thein vivoprotection required CD8 T cells. This provides clear experimental evidence that some multiply exposed, HIV-1-negative individuals havein vivoprotective immunity that is CD8 T cell-dependent. Understanding the mechanism of such protective immunity is critical to the design and testing of effective prophylactic vaccines and immunotherapeutic regimens.