bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of n-3 polyunsaturated fatty acids in brain: Modulation of rat brain gene expression by dietary n-3 fatty acids
Authors: Kitajka Klára , Puskás László G. , Zvara Ágnes , Hackler László , Barceló-Coblijn Gwendolyn , Yeo Young K. , Farkas Tibor
Published: July 2002

Rats were fed either a high linolenic acid (perilla oil) or high eicosapentaenoic + docosahexaenoic acid (fish oil) diet (8%), and the fatty acid and molecular species composition of ethanolamine phosphoglycerides was determined. Gene expression pattern resulting from the feeding of n-3 fatty acids also was studied. Perilla oil feeding, in contrast to fish oil feeding, was not reflected in total fatty acid composition of ethanolamine phosphoglycerides. Levels of the alkenylacyl subclass of ethanolamine phosphoglycerides increased in response to feeding. Similarly, levels of diacyl phosphatidylethanolamine molecular species containing docosahexaenoic acid (18:0/22:6) were higher in perilla-fed or fish oil-fed rat brains whereas those in ethanolamine plasmalogens remained unchanged. Because plasmalogen levels in the brains of rats fed a n-3 fatty acid-enriched diet increased, it is plausible, however, that docosahexaenoic acid taken up from the food or formed from linolenic acid was deposited in this phospholipid subclass. Using cDNA microarrays, 55 genes were found to be overexpressed and 47 were suppressed relative to controls by both dietary regimens. The altered genes included those controlling synaptic plasticity, cytosceleton and membrane association, signal transduction, ion channel formation, energy metabolism, and regulatory proteins. This effect seems to be independent of the chain length of fatty acids, but the n-3 structure appears to be important. Because n-3 polyunsaturated fatty acids have been shown to play an important role in maintaining normal mental functions and docosahexaenoic acid-containing ethanolamine phosphoglyceride (18:0/22:6) molecular species accumulated in response to n-3 fatty acid feeding, a casual relationship between the two events can be surmised.
TROSY-NMR reveals interaction between ERp57 and the tip of the calreticulin P-domain
Authors: Frickel Eva-Maria , Riek Roland , Jelesarov Ilian , Helenius Ari , Wüthrich Kurt , Ellgaard Lars
Published: July 2002

The lectin chaperone calreticulin (CRT) assists the folding and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). It interacts with ERp57, a thiol-disulfide oxidoreductase that promotes the formation of disulfide bonds in glycoproteins bound by CRT. Here, we investigated the interaction between CRT and ERp57 by using biochemical techniques and NMR spectroscopy. We found that ERp57 binds to the P-domain of calreticulin, an independently folding domain comprising residues 189–288. Isothermal titration calorimetry showed that the dissociation constant of the CRT(189–288)/ERp57 complex is (9.1 ± 3.0) × 10
−6
M at 8°C. Transverse relaxation-optimized NMR spectroscopy provided data on the thermodynamics and kinetics of the complex formation and on the structure of this 66.5-kDa complex. The NMR measurements yielded a value of (18 ± 5) × 10
−6
M at 20°C for the dissociation constant and a lower limit for the first-order exchange rate constant of
k
off
> 1,000 s
−1
at 20°C. Chemical shift mapping showed that interactions with ERp57 occur exclusively through amino acid residues in the polypeptide segment 225–251 of CRT(189–288), which forms the tip of the hairpin structure of this domain. These results are analyzed with regard to the functional mechanism of the CRT/ERp57 chaperone system.
The short interspersed repetitive element of
Trypanosoma cruzi
, SIRE, is part of VIPER, an unusual retroelement related to long terminal repeat retrotransposons
Authors: Vázquez Martín , Ben-Dov Claudia , Lorenzi Hernan , Moore Troy , Schijman Alejandro , Levin Mariano J.
Published: July 2002

The short interspersed repetitive element (SIRE) of
Trypanosoma cruzi
was first detected when comparing the sequences of loci that encode the
TcP2
β genes. It is present in about 1,500–3,000 copies per genome, depending on the strain, and it is distributed in all chromosomes. An initial analysis of SIRE sequences from 21 genomic fragments allowed us to derive a consensus nucleotide sequence and structure for the element, consisting of three regions (I, II, and III) each harboring distinctive features. Analysis of 158 transcribed SIREs demonstrates that the consensus is highly conserved. The sequences of 51 cDNAs show that SIRE is included in the 3′ end of several mRNAs, always transcribed from the sense strand, contributing the polyadenylation site in 63% of the cases. This study led to the characterization of VIPER (vestigial interposed retroelement), a 2,326-bp-long unusual retroelement. VIPER's 5′ end is formed by the first 182 bp of SIRE, whereas its 3′ end is formed by the last 220 bp of the element. Both SIRE moieties are connected by a 1,924-bp-long fragment that carries a unique ORF encoding a complete reverse transcriptase-RNase H gene whose 15 C-terminal amino acids derive from codons specified by SIRE's region II. The amino acid sequence of VIPER's reverse transcriptase-RNase H shares significant homology to that of long terminal repeat retrotransposons. The fact that SIRE and VIPER sequences are found only in the
T. cruzi
genome may be of relevance for studies concerning the evolution and the genome flexibility of this protozoan parasite.
Brain 5α-dihydroprogesterone and allopregnanolone synthesis in a mouse model of protracted social isolation
Authors: Dong Erbo , Matsumoto Kinzo , Uzunova Veska , Sugaya Ikuko , Takahata Hiroki , Nomura Hiroaki , Watanabe Hiroshi , Costa Erminio , Guidotti Alessandro
Published: July 2002

Allopregnanolone (ALLO), is a brain endogenous
neurosteroid that binds with high affinity to γ-aminobutyric acid
type A (GABA
A
) receptors and positively modulates the
action of GABA at these receptors. Unlike ALLO,
5α-dihydroprogesterone (5α-DHP) binds with high affinity to
intracellular progesterone receptors that regulate DNA transcription.
To investigate the physiological roles of ALLO and 5α-DHP synthesized
in brain, we have adopted a mouse model involving protracted social
isolation. In the frontal cortex of mice, socially isolated for 6
weeks, both neurosteroids were decreased by approximately 50%. After
administration of (17β)-17-(bis-1-methyl amino carbonyl)
androstane-3,5-diene-3-carboxylic acid (SKF105,111), an inhibitor of
the enzyme (5α-reductase Type I and II) that converts progesterone
into 5α-DHP, the ALLO and 5α-DHP content of frontal cortex of both
group-housed and socially isolated mice decreased exponentially to
10%–20% of control values in about 30 min. The fractional rate
constants (
k
h
−1
) of ALLO and 5α-DHP
decline multiplied by the ALLO and 5α-DHP concentrations at any given
steady-state estimate the rate of synthesis required to maintain that
steady state. After 6 weeks of social isolation, ALLO and 5α-DHP
biosynthesis rates were decreased to 30% of the values calculated in
group-housed mice. Moreover, in socially isolated mice, the expression
of 5α-reductase Type I mRNA and protein was approximately 50% lower
than in group-housed mice whereas 3α-hydroxysteroid oxidoreductase
mRNA expression was equal in the two groups. Protracted social
isolation in mice may provide a model to investigate whether 5α-DHP
by a genomic action, and ALLO by a nongenomic mechanism down-regulate
the action of drugs acting as agonists, partial agonists, or positive
allosteric modulators of the benzodiazepine recognition sites
expressed by GABA
A
receptors.
The crystal structure of rabbit phosphoglucose isomerase complexed with 5-phospho-
d
-arabinonohydroxamic acid
Authors: Arsenieva Diana , Hardré Renaud , Salmon Laurent , Jeffery Constance J.
Published: July 2002

Phosphoglucose isomerase (EC
5.3.1.9
) catalyzes the second step in glycolysis, the reversible isomerization of
d
-glucose 6-phosphate to
d
-fructose 6-phosphate. The reaction mechanism involves acid-base catalysis with proton transfer and proceeds through a
cis
-enediol(ate) intermediate. 5-Phospho-
d
-arabinonohydroxamic acid (5PAH) is a synthetic small molecule that resembles the reaction intermediate, differing only in that it has a nitrogen atom in place of C1. Hence, 5PAH is the best inhibitor of the isomerization reaction reported to date with a
K
i
of 2 × 10
−7
M. Here we report the crystal structure of rabbit phosphoglucose isomerase complexed with 5PAH at 1.9 Å resolution. The interaction of 5PAH with amino acid residues in the enzyme active site supports a model of the catalytic mechanism in which Glu-357 transfers a proton between C1 and C2 and Arg-272 helps stabilize the intermediate. It also suggests a mechanism for proton transfer between O1 and O2.
Requirement of Ca2+and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-γ
Authors: Nair Jayasree S. , DaFonseca Christopher J. , Tjernberg Agneta , Sun Wei , Darnell James E. , Chait Brian T. , Zhang J. Jillian
Published: July 2002

In response to IFN-γ, the latent cytoplasmic protein signal transducers and activators of transcription 1 (Stat1) becomes phosphorylated on Y701, dimerizes, and accumulates in the nucleus to activate transcription of IFN-γ-responsive genes. For maximal gene activation, S727 in the transcription activation domain of Stat1 also is inducibly phosphorylated by IFN-γ. We previously purified a group of nuclear proteins that interact specifically with the Stat1 transcription activation domain. In this report, we identified one of them as the multifunctional Ca2+/calmodulin-dependent kinase (CaMK) II. We demonstrate that IFN-γ mobilizes a Ca2+flux in cells and activates CaMKII. CaMKII can interact directly with Stat1 and phosphorylate Stat1 on S727in vitro. Inhibition of Ca2+flux or CaMKII results in a lack of S727 phosphorylation and Stat1-dependent gene activation, suggestingin vivophosphorylation of Stat1 S727 by CaMKII. Thus two different cellular signaling events, IFN-γ receptor occupation and Ca2+flux, are required for Stat1 to achieve maximal transcriptional activation through regulation of phosphorylation.
Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1
Authors: Michael Laura F. , Wu Zhidan , Cheatham R. Bentley , Puigserver Pere , Adelmant Guillaume , Lehman John J. , Kelly Daniel P. , Spiegelman Bruce M.
Published: July 2002

Muscle tissue is the major site for insulin-stimulated glucose
uptake
in vivo
, due primarily to the recruitment of the
insulin-sensitive glucose transporter (GLUT4) to the plasma membrane.
Surprisingly, virtually all cultured muscle cells express little or no
GLUT4. We show here that adenovirus-mediated expression of the
transcriptional coactivator PGC-1, which is expressed in muscle
in vivo
but is also deficient in cultured muscle cells,
causes the total restoration of GLUT4 mRNA levels to those observed
in vivo
. This increased GLUT4 expression correlates with
a 3-fold increase in glucose transport, although much of this protein
is transported to the plasma membrane even in the absence of insulin.
PGC-1 mediates this increased GLUT4 expression, in large part, by
binding to and coactivating the muscle-selective transcription factor
MEF2C. These data indicate that PGC-1 is a coactivator of MEF2C and can
control the level of endogenous GLUT4 gene expression in
muscle.
Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase of the
Triticum
/
Aegilops
complex and the evolutionary history of polyploid wheat
Authors: Huang Shaoxing , Sirikhachornkit Anchalee , Su Xiujuan , Faris Justin , Gill Bikram , Haselkorn Robert , Gornicki Piotr
Published: July 2002

The classic wheat evolutionary history is one of adaptive radiation of the diploid
Triticum
/
Aegilops
species (A, S, D), genome convergence and divergence of the tetraploid (
Triticum turgidum
AABB, and
Triticum timopheevii
AAGG) and hexaploid (
Triticum aestivum
, AABBDD) species. We analyzed
Acc-1
(plastid acetyl-CoA carboxylase) and
Pgk-1
(plastid 3-phosphoglycerate kinase) genes to determine phylogenetic relationships among
Triticum
and
Aegilops
species of the wheat lineage and to establish the timeline of wheat evolution based on gene sequence comparisons.
Triticum urartu
was confirmed as the A genome donor of tetraploid and hexaploid wheat. The A genome of polyploid wheat diverged from
T. urartu
less than half a million years ago (MYA), indicating a relatively recent origin of polyploid wheat. The D genome sequences of
T. aestivum
and
Aegilops tauschii
are identical, confirming that
T. aestivum
arose from hybridization of
T. turgidum
and
Ae. tauschii
only 8,000 years ago. The diploid
Triticum
and
Aegilops
progenitors of the A, B, D, G, and S genomes all radiated 2.5–4.5 MYA. Our data suggest that the
Acc-1
and
Pgk-1
loci have different histories in different lineages, indicating genome mosaicity and significant intraspecific differentiation. Some loci of the S genome of
Aegilops speltoides
and the G genome of
T. timophevii
are closely related, suggesting the same origin of some parts of their genomes. None of the
Aegilops
genomes analyzed is a close relative of the B genome, so the diploid progenitor of the B genome remains unknown.
Renewable and nonrenewable resources: Amino acid turnover and allocation to reproduction in Lepidoptera
Authors: O'Brien Diane M. , Fogel Marilyn L. , Boggs Carol L.
Published: July 2002

The allocation of nutritional resources to reproduction in animals is a complex process of great evolutionary significance. We use compound-specific stable isotope analysis of carbon (GC/combustion/isotope ratio MS) to investigate the dietary sources of egg amino acids in a nectar-feeding hawkmoth. Previous work suggests that the nutrients used in egg manufacture fall into two classes: those that are increasingly synthesized from adult dietary sugar over a female's lifetime (renewable resources), and those that remain exclusively larval in origin (nonrenewable resources). We predict that nonessential and essential amino acids correspond to these nutrient classes and test this prediction by analyzing egg amino acids from females fed isotopically distinct diets as larvae and as adults. The results demonstrate that essential egg amino acids originate entirely from the larval diet. In contrast, nonessential egg amino acids were increasingly synthesized from adult dietary sugars, following a turnover pattern across a female's lifetime. This study demonstrates that female Lepidoptera can synthesize a large fraction of egg amino acids from nectar sugars, using endogenous sources of nitrogen. However, essential amino acids derive only from the larval diet, placing an upper limit on the use of adult dietary resources to enhance reproductive success.
A minimal system allowing tubulation with molecular motors pulling on giant liposomes
Authors: Roux Aurélien , Cappello Giovanni , Cartaud Jean , Prost Jacques , Goud Bruno , Bassereau Patricia
Published: July 2002

The elucidation of physical and molecular mechanisms by which a membrane tube is generated from a membrane reservoir is central to the understanding of the structure and dynamics of intracellular organelles and of transport intermediates in eukaryotic cells. Compelling evidence exists that molecular motors of the dynein and kinesin families are involved in the tubulation of organelles. Here, we show that lipid giant unilamellar vesicles (GUVs), to which kinesin molecules have been attached by means of small polystyrene beads, give rise to membrane tubes and to complex tubular networks when incubated
in vitro
with microtubules and ATP. Similar tubes and networks are obtained with GUVs made of purified Golgi lipids, as well as with Golgi membranes. No tube formation was observed when kinesins were directly bound to the GUV membrane, suggesting that it is critical to distribute the load on both lipids and motors by means of beads. A kinetic analysis shows that network growth occurs in two phases: a phase in which membrane-bound beads move at the same velocity than free beads, followed by a phase in which the tube growth rate decreases and strongly fluctuates. Our work demonstrates that the action of motors bound to a lipid bilayer is sufficient to generate membrane tubes and opens the way to well controlled experiments aimed at the understanding of basic mechanisms in intracellular transport.
A selective peroxisome proliferator-activated receptor δ agonist promotes reverse cholesterol transport
Authors: Oliver William R. , Shenk Jennifer L. , Snaith Mike R. , Russell Caroline S. , Plunket Kelli D. , Bodkin Noni L. , Lewis Michael C. , Winegar Deborah A. , Sznaidman Marcos L. , Lambert Millard H. , Xu H. Eric , Sternbach Daniel D. , Kliewer Steven A. , Hansen Barbara C. , Willson Timothy M.
Published: July 2002

The peroxisome proliferator-activated receptors (PPARs) are dietary
lipid sensors that regulate fatty acid and carbohydrate metabolism. The
hypolipidemic effects of the fibrate drugs and the antidiabetic effects
of the glitazone drugs in humans are due to activation of the α
(NR1C1) and γ (NR1C3) subtypes, respectively. By contrast, the
therapeutic potential of the δ (NR1C2) subtype is unknown, due in
part to the lack of selective ligands. We have used combinatorial
chemistry and structure-based drug design to develop a potent and
subtype-selective PPARδ agonist, GW501516. In macrophages,
fibroblasts, and intestinal cells, GW501516 increases expression of the
reverse cholesterol transporter ATP-binding cassette A1 and induces
apolipoprotein A1-specific cholesterol efflux. When dosed to
insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a
dramatic dose-dependent rise in serum high density lipoprotein
cholesterol while lowering the levels of small-dense low density
lipoprotein, fasting triglycerides, and fasting insulin. Our results
suggest that PPARδ agonists may be effective drugs to increase
reverse cholesterol transport and decrease cardiovascular disease
associated with the metabolic syndrome X.
Disrupting evolutionary processes: The effect of habitat fragmentation on collared lizards in the Missouri Ozarks
Authors: Templeton Alan R. , Robertson Robert J. , Brisson Jennifer , Strasburg Jared
Published: July 2002

Humans affect biodiversity at the genetic, species, community, and
ecosystem levels. This impact on genetic diversity is critical, because
genetic diversity is the raw material of evolutionary change, including
adaptation and speciation. Two forces affecting genetic variation are
genetic drift (which decreases genetic variation within but increases
genetic differentiation among local populations) and gene flow (which
increases variation within but decreases differentiation among local
populations). Humans activities often augment drift and diminish gene
flow for many species, which reduces genetic variation in local
populations and prevents the spread of adaptive complexes outside their
population of origin, thereby disrupting adaptive processes both
locally and globally within a species. These impacts are illustrated
with collared lizards (
Crotaphytus collaris
) in the
Missouri Ozarks. Forest fire suppression has reduced habitat and
disrupted gene flow in this lizard, thereby altering the balance toward
drift and away from gene flow. This balance can be restored by managed
landscape burns. Some have argued that, although human-induced
fragmentation disrupts adaptation, it will also ultimately produce new
species through founder effects. However, population genetic theory and
experiments predict that most fragmentation events caused by human
activities will facilitate not speciation, but local extinction.
Founder events have played an important role in the macroevolution of
certain groups, but only when ecological opportunities are expanding
rather than contracting. The general impact of human activities on
genetic diversity disrupts or diminishes the capacity for adaptation,
speciation, and macroevolutionary change. This impact will ultimately
diminish biodiversity at all levels.
Multiple maternal origins and weak phylogeographic structure in domestic goats
Authors: Luikart Gordon , Gielly Ludovic , Excoffier Laurent , Vigne Jean-Denis , Bouvet Jean , Taberlet Pierre
Published: July 2002

Domestic animals have played a key role in human history. Despite
their importance, however, the origins of most domestic species remain
poorly understood. We assessed the phylogenetic history and population
structure of domestic goats by sequencing a hypervariable segment (481
bp) of the mtDNA control region from 406 goats representing 88 breeds
distributed across the Old World. Phylogeographic analysis revealed
three highly divergent goat lineages (estimated divergence >200,000
years ago), with one lineage occurring only in eastern and southern
Asia. A remarkably similar pattern exists in cattle, sheep, and pigs.
These results, combined with recent archaeological findings, suggest
that goats and other farm animals have multiple maternal origins with a
possible center of origin in Asia, as well as in the Fertile Crescent.
The pattern of goat mtDNA diversity suggests that all three lineages
have undergone population expansions, but that the expansion was
relatively recent for two of the lineages (including the Asian
lineage). Goat populations are surprisingly less genetically structured
than cattle populations. In goats only ≈10% of the mtDNA variation
is partitioned among continents. In cattle the amount is ≥50%. This
weak structuring suggests extensive intercontinental transportation of
goats and has intriguing implications about the importance of goats in
historical human migrations and commerce.
The identification of functional modules from the genomic association of genes
Authors: Snel Berend , Bork Peer , Huynen Martijn A.
Published: July 2002

By combining the pairwise interactions between proteins, as predicted by the conserved co-occurrence of their genes in operons, we obtain protein interaction networks. Here we study the properties of such networks to identify functional modules: sets of proteins that together are involved in a biological process. The complete network contains 3,033 orthologous groups of proteins in 38 genomes. It consists of one giant component, containing 1,611 orthologous groups, and of 516 small disjointed clusters that, on average, contain only 2.7 orthologous groups. These small clusters have a homogeneous functional composition and thus represent functional modules in themselves. Analysis of the giant component reveals that it is a scale-free, small-world network with a high degree of local clustering (C = 0.6). It consists of locally highly connected subclusters that are connected to each other by linker proteins. The linker proteins tend to have multiple functions, or are involved in multiple processes and have an above average probability of being essential. By splitting up the giant component at these linker proteins, we identify 265 subclusters that tend to have a homogeneous functional composition. The rare functional inhomogeneities in our subclusters reflect the mixing of different types of (molecular) functions in a single cellular process, exemplified by subclusters containing both metabolic enzymes as well as the transcription factors that regulate them. Comparative genome analysis, thus, allows identification of a level of functional interaction between that of pairwise interactions, and of the complete genome.
A syntactic specialization for Broca's area
Authors: Embick David , Marantz Alec , Miyashita Yasushi , O'Neil Wayne , Sakai Kuniyoshi L.
Published: July 2002

Despite numerous aphasia and functional imaging studies, the exact correlation between cortical language areas and subcomponents of the linguistic system has not been established. Here, we used functional MRI to identify cortical areas specifically involved in syntactic processing. An experimental design contrasted sentences containing grammatical errors with sentences containing spelling errors. The ungrammatical sentences produced more activation in cortical language areas than did the sentences with spelling errors, and the difference in activation was significantly greater in Broca's area than in Wernicke's area or in the angular gyrus/supramarginal gyrus. The present findings provide direct evidence of a syntactic specialization for Broca's area and establish the existence of distinct modules for our knowledge of language.
Beyond re-membering: Phantom sensations of congenitally absent limbs
Authors: Brugger Peter , Kollias Spyros S. , Müri René M. , Crelier Gérard , Hepp-Reymond Marie-Claude , Regard Marianne
Published: July 2002

Phantom limbs are traditionally conceptualized as the phenomenal persistence of a body part after deafferentation. Previous clinical observations of subjects with phantoms of congenitally absent limbs are not compatible with this view, but, in the absence of experimental work, the neural basis of such “aplasic phantoms” has remained enigmatic. In this paper, we report a series of behavioral, imaging, and neurophysiological experiments with a university-educated woman born without forearms and legs, who experiences vivid phantom sensations of all four limbs. Visuokinesthetic integration of tachistoscopically presented drawings of hands and feet indicated an intact somatic representation of these body parts. Functional magnetic resonance imaging of phantom hand movements showed no activation of primary sensorimotor areas, but of premotor and parietal cortex bilaterally. Movements of the existing upper arms produced activation expanding into the hand territories deprived of afferences and efferences. Transcranial magnetic stimulation of the sensorimotor cortex consistently elicited phantom sensations in the contralateral fingers and hand. In addition, premotor and parietal stimulation evoked similar phantom sensations, albeit in the absence of motor evoked potentials in the stump. These data indicate that body parts that have never been physically developed can be represented in sensory and motor cortical areas. Both genetic and epigenetic factors, such as the habitual observation of other people moving their limbs, may contribute to the conscious experience of aplasic phantoms.
Mayolenes: Labile defensive lipids from the glandular hairs of a caterpillar (
Pieris rapae
)
Authors: Smedley Scott R. , Schroeder Frank C. , Weibel Douglas B. , Meinwald Jerrold , Lafleur Katie A. , Renwick J. Alan , Rutowski Ronald , Eisner Thomas
Published: July 2002

Larvae of the European cabbage butterfly,
Pieris rapae
(Pieridae)
,
are beset with glandular hairs, bearing droplets of a clear oily secretion at their tip. The fluid consists primarily of a series of chemically labile, unsaturated lipids, the mayolenes, which are derived from 11-hydroxylinolenic acid. In bioassays with the ant
Crematogaster lineolata,
the secretion was shown to be potently deterrent, indicating that the fluid plays a defensive role in nature.
The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine-sulfenic acid derivative
Authors: Fuangthong Mayuree , Helmann John D.
Published: July 2002

Reactive oxygen species induce the expression of detoxification and repair genes critical for life in an aerobic environment. Bacterial factors that sense reactive oxygen species use either thiol-disulfide exchange reactions (OxyR, RsrA) or redox labile 2Fe–2S clusters (SoxR). We demonstrate that the reduced form of
Bacillus subtilis
OhrR binds cooperatively to two adjacent inverted repeat sequences in the
ohrA
control region and thereby represses transcription. In the presence of organic hydroperoxides, OhrR is inactivated by the reversible oxidation of a single conserved cysteine residue to the corresponding cysteine-sulfenic acid, and perhaps to higher oxidation states.
Pro-apoptotic gene expression mediated by the p38 mitogen-activated protein kinase signal transduction pathway
Authors: De Zutter Gerard S. , Davis Roger J.
Published: July 2002

Neurotrophic factor deprivation causes apoptosis by a
mechanism that requires macromolecular synthesis. This fact suggests
that gene expression is necessary to achieve cell death. To identify
mRNA that is expressed in apoptotic cells we used subtractive
hybridization with cDNA prepared from neuronal pheochromocytoma cells.
Monoamine oxidase (MAO) expression was increased in cells during nerve
growth factor withdrawal-induced apoptosis. The increased
apoptosis and induction of MAO was prevented by inhibition of
the p38 mitogen-activated protein (MAP) kinase pathway. MAO may
contribute to the apoptotic process because inhibition of MAO
activity suppressed cell death. Together, these data indicate that MAO
may be a target of pro-apoptotic signal transduction by the p38
MAP kinase pathway.
Blood flow and oxygen delivery to human brain during functional activity: Theoretical modeling and experimental data
Authors: Mintun Mark A. , Lundstrom Brian N. , Snyder Abraham Z. , Vlassenko Andrei G. , Shulman Gordon L. , Raichle Marcus E.
Published: July 2002

Coupling of cerebral blood flow (CBF) and cerebral metabolic
rate for oxygen (CMRO
2
) in physiologically activated brain
states remains the subject of debates. Recently it was suggested that
CBF is tightly coupled to oxidative metabolism in a nonlinear fashion.
As part of this hypothesis, mathematical models of oxygen delivery to
the brain have been described in which disproportionately large
increases in CBF are necessary to sustain even small increases in
CMRO
2
during activation. We have explored the coupling of
CBF and oxygen delivery by using two complementary methods. First, a
more complex mathematical model was tested that differs from those
recently described in that no assumptions were made regarding tissue
oxygen level. Second, [
15
O] water CBF positron emission
tomography (PET) studies in nine healthy subjects were conducted during
states of visual activation and hypoxia to examine the relationship of
CBF and oxygen delivery. In contrast to previous reports, our model
showed adequate tissue levels of oxygen could be maintained without the
need for increased CBF or oxygen delivery. Similarly, the PET studies
demonstrated that the regional increase in CBF during visual activation
was not affected by hypoxia. These findings strongly indicate that the
increase in CBF associated with physiological activation is regulated
by factors other than local requirements in oxygen.
Identification of 86 candidates for small non-messenger RNAs from the archaeon
Archaeoglobus
fulgidus
Authors: Tang Thean-Hock , Bachellerie Jean-Pierre , Rozhdestvensky Timofey , Bortolin Marie-Line , Huber Harald , Drungowski Mario , Elge Thorsten , Brosius Jürgen , Hüttenhofer Alexander
Published: July 2002

In a specialized cDNA library from the archaeon
Archaeoglobus fulgidus
we have identified a total of 86 different expressed RNA sequences potentially encoding previously uncharacterized
s
mall
n
on-
m
essenger
RNA
(snmRNA) species. Ten of these RNAs resemble eukaryotic small nucleolar RNAs, which guide rRNA 2′-
O
-methylations (C/D-box type) and pseudouridylations (H/ACA-box type). Thereby, we identified four candidates for H/ACA small RNAs in an archaeal species that are predicted to guide a total of six rRNA pseudouridylations. Furthermore, we have verified the presence of the six predicted pseudouridines experimentally. We demonstrate that 22 snmRNAs are transcribed from a family of short tandem repeats conserved in most archaeal genomes and shown previously to be potentially involved in replicon partitioning. In addition, four snmRNAs derived from the rRNA operon of
A. fulgidus
were identified and shown to be generated by a splicing/processing pathway of pre-rRNAs. The remaining 50 RNAs could not be assigned to a known class of snmRNAs because of the lack of known structure and/or sequence motifs. Regarding their location on the genome, only nine were located in intergenic regions, whereas 33 were complementary to an ORF, five were overlapping an ORF, and three were derived from the sense orientation within an ORF. Our study further supports the importance of snmRNAs in all three domains of life.
Rad52 forms DNA repair and recombination centers during S phase
Authors: Lisby Michael , Rothstein Rodney , Mortensen Uffe H.
Published: July 2002

Maintenance of genomic integrity and stable transmission of genetic
information depend on a number of DNA repair processes. Failure to
faithfully perform these processes can result in genetic alterations
and subsequent development of cancer and other genetic diseases. In the
eukaryote
Saccharomyces cerevisiae
, homologous
recombination is the major pathway for repairing DNA double-strand
breaks. The key role played by Rad52 in this pathway has been
attributed to its ability to seek out and mediate annealing of
homologous DNA strands. In this study, we find that
S.
cerevisiae
Rad52 fused to green fluorescent protein (GFP) is
fully functional in DNA repair and recombination. After induction of
DNA double-strand breaks by γ
-
irradiation, meiosis, or
the
HO
endonuclease, Rad52-GFP relocalizes from a
diffuse nuclear distribution to distinct foci. Interestingly, Rad52
foci are formed almost exclusively during the S phase of mitotic cells,
consistent with coordination between recombinational repair and DNA
replication. This notion is further strengthened by the dramatic
increase in the frequency of Rad52 focus formation observed in a
pol12
-
100
replication mutant and a
mec1
DNA damage checkpoint mutant. Furthermore, our data
indicate that each Rad52 focus represents a center of recombinational
repair capable of processing multiple DNA lesions.
Dopamine receptor regulating factor, DRRF: A zinc finger transcription factor
Authors: Hwang Cheol Kyu , D'Souza Ursula M. , Eisch Amelia J. , Yajima Shunsuke , Lammers Claas-Hinrich , Yang Young , Lee Sang-Hyeon , Kim Yong-Man , Nestler Eric J. , Mouradian M. Maral
Published: July 2002

Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D
1A
and D
2
dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors.
In vivo
, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain.
Single-strand interruptions in replicating chromosomes cause double-strand breaks
Authors: Kuzminov Andrei
Published: July 2002

Replication-dependent chromosomal breakage suggests that replication forks occasionally run into nicks in template DNA and collapse, generating double-strand ends. To model replication fork collapse
in vivo
, I constructed phage λ chromosomes carrying the nicking site of M13 bacteriophage and infected with these substrates
Escherichia coli
cells, producing M13 nicking enzyme. I detected double-strand breaks at the nicking sites in λ DNA purified from these cells. The double-strand breakage depends on (
i
) the presence of the nicking site; (
ii
) the production of the nicking enzyme; and (
iii
) replication of the nick-containing chromosome. Replication fork collapse at nicks in template DNA explains diverse phenomena, including eukaryotic cell killing by DNA topoisomerase inhibitors and inviability of recombination-deficient vertebrate cell lines.
Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55
Gag
Authors: VerPlank Lynn , Bouamr Fadila , LaGrassa Tracy J. , Agresta Beth , Kikonyogo Alexandra , Leis Jonathan , Carter Carol A.
Published: July 2002

Ubiquitination appears to be involved in virus particle release from infected cells. Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles. Here we report that the p6 region in the Pr55
Gag
structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins. Tsg101 was found to interact with Gag in (
i
) a yeast two-hybrid assay, (
ii
)
in vitro
coimmunoprecipitation by using purified Pr55
Gag
and rabbit reticulocyte lysate-synthesized Tsg101, and (
iii
)
in vivo
in the cytoplasm of COS cells transfected with
gag
. The PTAPP motif [or late (L) domain] within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55
Gag
. The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6. Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein. The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus.
Nucleotide binding by the erythrocyte transglutaminase/G
h
protein, probed with fluorescent analogs of GTP and GDP
Authors: Murthy S. N. Prasanna , Lorand Laszlo
Published: July 2002

GTP is known to be a potent inhibitor of the protein crosslinking activity of transglutaminase (TG), probably the most abundant G protein in the human red cell. Nucleotide binding to TG was examined by fluorescence spectroscopy and anisotropy in mixtures of TG with methylanthraniloyl analogs of GTP and GDP. A characteristic feature was the appearance of a major energy transfer band (λ
exc, max
= 290 nm, λ
em
= 444 nm) from protein tryptophans to the bound nucleotides. Quenching of the bound fluorophore (λ
exc
= 360 nm, λ
em
= 444 nm) by acrylamide was barely different from that of free ligand. However, major changes were observed in anisotropy, which was used to demonstrate a facile exchange between bound and free nucleotides and to evaluate affinity constants for the binding of methylanthraniloyl GTP and GDP to TG.
How αβ T cells deal with induced TCRα ablation
Authors: Polic Bojan , Kunkel Desiree , Scheffold Alexander , Rajewsky Klaus
Published: July 2002

On deletion of the gene encoding the constant region of the T cell antigen receptor (TCR)α chain in mature T cells by induced Cre-mediated recombination, the cells lose most of their TCR from the cell surface within 7–10 days, but minute amounts of surface-bound TCRβ chains are retained for long periods of time. In a situation in which cellular influx from the thymus is blocked, TCR-deficient naïve T cells decay over time, the decay rates being faster for CD8+cells (t1/2≈ 16 days) than for CD4+cells (t1/2≈ 46 days). TCR+naïve cells are either maintained (CD8+) or decay more slowly (CD4+;t1/2≈ 78 days.) Numbers of TCR-deficient memory T cells decline very slowly (CD8+cells;t1/2≈ 52 days) or not at all (CD4+cells), but at the population level, these cells fail to expand as their TCR+counterparts do. Together with earlier data on T cell maintenance in environments lacking appropriate major histocompatibility complex antigens, these data argue against the possibility that spontaneous ligand-independent signaling by the αβTCR contributes significantly to T-cell homeostasis.
Avian-to-human transmission of H9N2 subtype influenza A viruses: Relationship between H9N2 and H5N1 human isolates
Authors: Lin Y. P. , Shaw M. , Gregory V. , Cameron K. , Lim W. , Klimov A. , Subbarao K. , Guan Y. , Krauss S. , Shortridge K. , Webster R. , Cox N. , Hay A.
Published: July 2002

In 1997, 18 cases of influenza in Hong Kong (bird flu) caused by a novel H5N1 (chicken) virus resulted in the deaths of six individuals and once again raised the specter of a potentially devastating influenza pandemic. Slaughter of the poultry in the live bird markets removed the source of infection and no further human cases of H5N1 infection have occurred. In March 1999, however, a new pandemic threat appeared when influenza A H9N2 viruses infected two children in Hong Kong. These two virus isolates are similar to an H9N2 virus isolated from a quail in Hong Kong in late 1997. Although differing in their surface hemagglutinin and neuraminidase components, a notable feature of these H9N2 viruses is that the six genes encoding the internal components of the virus are similar to those of the 1997 H5N1 human and avian isolates. This common feature emphasizes the apparent propensity of avian viruses with this genetic complement to infect humans and highlights the potential for the emergence of a novel human pathogen.
Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection
Authors: Schweitzer Barry , Wiltshire Steven , Lambert Jeremy , O'Malley Shawn , Kukanskis Kari , Zhu Zhengrong , Kingsmore Stephen F. , Lizardi Paul M. , Ward David C.
Published: July 2002

We describe an adaptation of the rolling circle amplification (RCA) reporter system for the detection of protein Ags, termed “immunoRCA.” In immunoRCA, an oligonucleotide primer is covalently attached to an Ab; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, amplification results in a long DNA molecule containing hundreds of copies of the circular DNA sequence that remain attached to the Ab and that can be detected in a variety of ways. Using immunoRCA, analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays in ELISA and microparticle formats. The signal amplification afforded by immunoRCA also enabled immunoassays to be carried out in microspot and microarray formats with exquisite sensitivity. When Ags are present at concentrations down to fM levels, specifically bound Abs can be scored by counting discrete fluorescent signals arising from individual Ag–Ab complexes. Multiplex immunoRCA also was demonstrated by accurately quantifying Ags mixed in different ratios in a two-color, single-molecule-counting assay on a glass slide. ImmunoRCA thus combines high sensitivity and a very wide dynamic range with an unprecedented capability for single molecule detection. This Ag-detection method is of general applicability and is extendable to multiplexed immunoassays that employ a battery of different Abs, each labeled with a unique oligonucleotide primer, that can be discriminated by a color-coded visualization system. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis.
The Sec6/8 complex in mammalian cells: Characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells
Authors: Matern Hugo T. , Yeaman Charles , Nelson W. James , Scheller Richard H.
Published: July 2002

The yeast exocyst complex (also called Sec6/8 complex in higher
eukaryotes) is a multiprotein complex essential for targeting exocytic
vesicles to specific docking sites on the plasma membrane. It is
composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70
and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian
orthologues for seven of these proteins have been described and here we
report the cloning and initial characterization of the remaining
subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with
yeast Sec3p, interacts in the two-hybrid system with other subunits of
the complex (Sec5 and Sec8), and is expressed in almost all tissues
tested. In yeast, Sec3p has been proposed to be a spatial landmark for
polarized secretion (1), and its localization depends on its
interaction with Rho1p (2). We demonstrate here that hSec3 lacks the
potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic.
Green fluorescent protein (GFP)-fusions of nearly every subunit of the
mammalian Sec6/8 complex were expressed in Madin–Darby canine kidney
(MDCK) cells, but they failed to assemble into a complex with
endogenous proteins and localized in the cytosol. Of the
subunits tested, only GFP-Exo70 localized to lateral membrane sites of
cell–cell contact when expressed in MDCK cells. Cells overexpressing
GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70
targeting interaction is critical for normal development of polarized
epithelial cells.
The see-through medaka: A fish model that is transparent throughout life
Authors: Wakamatsu Yuko , Pristyazhnyuk Sergey , Kinoshita Masato , Tanaka Minoru , Ozato Kenjiro
Published: July 2002

The see-through medaka is a vertebrate model with a transparent body in the adult stage, as well as during the embryonic stages, that was generated from a small laboratory fish, medaka (
Oryzias latipes
). In this fish model, most of the pigments are genetically removed from the entire body by a combination of recessive alleles at four loci. The main internal organs, namely, heart, spleen, blood vessels, liver, gut, gonads, kidney, brain, spinal cord, lens, air bladder, and gills, in living adult fish are visible to the naked eye or with a simple stereoscopic microscope. This fish is healthy and fertile. A transgenic see-through medaka was produced by using the green fluorescent protein (
GFP
) gene fused to the regulatory regions of the medaka
vasa
gene, in which germ cell-specific expression of
GFP
was visualized. The fluorescent tag also efficiently improved visibility of gonadal tissues. The process of oocyte maturation in the ovary was monitored by repeated observations from the outside of the body during one spawning cycle in the same living females of the transgenic see-through stock. The see-through medaka will provide an opportunity for noninvasive studies of morphological and molecular events that occur in internal organs in the later stages of life.
Glutamate-induced transient modification of the postsynaptic density
Authors: Dosemeci Ayse , Tao-Cheng Jung-Hwa , Vinade Lucia , Winters Christine A. , Pozzo-Miller Lucas , Reese Thomas S.
Published: July 2002

Depolarization of rat hippocampal neurons with a high concentration of external potassium induces a thickening of postsynaptic densities (PSDs) within 1.5–3 min. After high-potassium treatment, PSDs thicken 2.1-fold in cultured neurons and 1.4-fold in hippocampal slices compared with their respective controls. Thin-section immunoelectron microscopy of hippocampal cultures indicates that at least part of the observed thickening of PSDs can be accounted for by an accumulation of Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) on their cytoplasmic faces. Indeed, PSD-associated gold label for CaMKII increases 5-fold after depolarization with potassium. The effects of high-potassium treatment on the composition and structure of the PSDs are mimicked by direct application of glutamate. In cultures, glutamate-induced thickening of PSDs and the accumulation of CaMKII on PSDs are reversed within 5 min of removal of glutamate and Ca
2+
from the extracellular medium. These results suggest that PSDs are dynamic structures whose thickness and composition are subject to rapid and transient changes during synaptic activity.