bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated retinoic acid receptor β
4
protein in human breast tumor cells with nuclear and cytoplasmic localization
Authors: Sommer Karen M. , Chen Lucinda I. , Treuting Piper M. , Smith Lynne T. , Swisshelm Karen
Published: July 2002

The transcription factor retinoic acid receptor β
2
(RARβ
2
) is a potent inhibitor of breast cancer cells
in vitro
, and studies suggest that RARβ expression is lost in primary breast cancer. Although RARβ
2
is selectively down-regulated at the mRNA level in breast tumor cells, we show that expression of an RARβ protein is elevated in five of five breast tumor cell lines relative to normal human mammary epithelial cells. Subsequent analysis identified this protein as the translation product of the human RARβ
4
transcript. Unlike the previously characterized mouse RARβ
4
isoform, the human RARβ
4
retains only half of a DNA-binding domain and lacks a ligand-independent transactivation domain at its N terminus. The RARβ
4
protein localizes to the cytoplasm and to subnuclear compartments that resemble nuclear bodies. The structure and preliminary characterizations of human RARβ
4
, coupled with the observation that its expression is greatly elevated in breast tumor cell lines, support the hypothesis that RARβ
4
functions as a dominant-negative repressor of RAR-mediated growth suppression.
Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons
Authors: Chalecka-Franaszek Elzbieta , Chuang De-Maw
Published: July 2002

This report describes a modulatory action of lithium and glutamate on the activity of serine/threonine kinase Akt-1. Lithium is most commonly used to treat bipolar disorder, but the mechanism of its therapeutic action remains unknown. We have recently demonstrated that lithium protects against glutamate-induced excitotoxicity in cultured brain neurons and in an animal model of cerebral ischemia. This study was undertaken to investigate the role of Akt-1, activated by the phosphatidylinositol 3-kinase (PI 3-K) signaling pathway, in mediating glutamate excitotoxicity and lithium protection in cerebellar granule cells. High levels of phosphorylation and activity of Akt-1 were detected in cerebellar neurons cultured in the presence of serum. Protracted treatment with selective PI 3-K inhibitors, wortmannin and LY294002, abolished Akt-1 activity and induced neuronal death that could be reduced by long-term lithium pretreatment. Exposure of cells to glutamate induced a rapid and reversible loss of Akt-1 phosphorylation and kinase activity. These effects were closely correlated with excitotoxicity and caspase 3 activation and were prevented by phosphatase inhibitors, okadaic acid and caliculin A. Long-term lithium pretreatment suppressed glutamate-induced loss of Akt-1 activity and accelerated its recovery toward the control levels. Lithium treatment alone induced rapid increase in PI 3-K activity, and Akt-1 phosphorylation with accompanying kinase activation, which was blocked by PI 3-K inhibitors. Lithium also increased the phosphorylation of glycogen synthase kinase-3 (GSK-3), a downstream physiological target of Akt. Thus, modulation of Akt-1 activity appears to play a key role in the mechanism of glutamate excitotoxicity and lithium neuroprotection.
Genetic benefits enhance the reproductive success of polyandrous females
Authors: Newcomer Scott D. , Zeh Jeanne A. , Zeh David W.
Published: July 2002

Although it is generally accepted that females can gain material benefits by mating with more than one male, the proposal that polyandry provides genetic benefits remains controversial, largely because direct experimental support is lacking. Here, we report the results of a study testing for genetic benefits to polyandry in the pseudoscorpionCordylochernes scorpioides. In an experiment that controlled for male mating experience and the number of spermatophores accepted by a female, twice-mated females received either one sperm-packet from each of two different males (the “DM” treatment) or two sperm-packets from a single male (the same male or “SM” treatment). Over their lifetime, DM females gave birth to 32% more offspring than did SM females, primarily because of a significantly reduced rate of spontaneous abortion. This result could not be attributed to male infertility nor to lack of sexual receptivity in males paired with previous mates. Spermatophore and sperm numbers did not differ between males presented with a previous mate and males paired with a new female. Because SM and DM females received the same quantity of ejaculate, it was possible to eliminate material benefits as a contributor to the enhanced reproductive success of DM females. The reduction in embryo failure rate achieved by DM females is most consistent with the genetic incompatibility avoidance hypothesis, i.e., that polyandry enables females to exploit postcopulatory mechanisms for reducing the risk and/or cost of fertilization by genetically incompatible sperm. This study, which rigorously controlled for material benefits and excluded inbreeding effects, demonstrates that polyandry provides genetic benefits that significantly enhance female lifetime reproductive success.
Asparagine and glutamine rotamers:
B
-factor cutoff and correction of amide flips yield distinct clustering
Authors: Lovell Simon C. , Word J. Michael , Richardson Jane S. , Richardson David C.
Published: July 2002

Previous rotamer libraries showed little significant clustering for asparagine χ
2
or glutamine χ
3
values, but none of those studies corrected amide orientations or omitted disordered side chains. The current survey used 240 proteins at ≤1.7 Å resolution with <50% homology and <30 clashes per thousand atoms (atomic overlap ≥0.4 Å). All H atoms were added and optimized, and amide orientation was flipped by 180° if required by H bonding or atomic clashes. A side chain was included only if its amide orientation was clearly determined and if no atom had a
B
factor ≥40, alternate conformation, or severe clash; that selection process yielded 1,490 Asn and 863 Gln side chains. Clear clustering was observed for Asn χ
2
and Gln χ
3
(except when Gln χ
2
is
trans
). For Gln, five major and four minor rotamers cover 87% of examples. For Asn, there are seven backbone-independent rotamers covering 94% of examples plus rotamers specified for strictly α-helical, β, and left-handed (+φ) Asn. Although the strongest influence on χ angles is avoidance of atomic clashes (especially with the NH
2
hydrogens), some Asn or Gln rotamers are influenced by favorable van der Waals contacts and others by specific local H-bond patterns.
Deficit of
in vivo
mitochondrial ATP production in patients with Friedreich ataxia
Authors: Lodi Raffaele , Cooper J. Mark , Bradley Jane L. , Manners David , Styles Peter , Taylor Doris J. , Schapira Anthony H. V.
Published: July 2002

Friedreich ataxia (FRDA), the most common of the inherited ataxias, is an autosomal recessive degenerative disorder, characterized clinically by onset before the age of 25 of progressive gait and limb ataxia, absence of deep tendon reflexes, extensor plantar responses, and loss of position and vibration sense in the lower limbs. FRDA is caused by a GAA triplet expansion in the first intron of the
FRDA
gene on chromosome 9q13 in 97% of patients. The
FRDA
gene encodes a widely expressed 210-aa protein, frataxin, which is located in mitochondria and is severely reduced in FRDA patients. Frataxin function is still unknown but the knockout of the yeast frataxin homologue gene (
YFH1
) showed a severe defect of mitochondrial respiration and loss of mtDNA associated with elevated intramitochondrial iron. Here we report
in vivo
evidence of impaired mitochondrial respiration in skeletal muscle of FRDA patients. Using phosphorus magnetic resonance spectroscopy we demonstrated a maximum rate of muscle mitochondrial ATP production (
V
max
) below the normal range in all 12 FRDA patients and a strong negative correlation between mitochondrial
V
max
and the number of GAA repeats in the smaller allele. Our results show that FRDA is a nuclear-encoded mitochondrial disorder affecting oxidative phosphorylation and give a rationale for treatments aimed to improve mitochondrial function in this condition.
Direct molecular force measurements of multiple adhesive interactions between cadherin ectodomains
Authors: Sivasankar S. , Brieher W. , Lavrik N. , Gumbiner B. , Leckband D.
Published: July 2002

Direct-force measurements of the interactions between recombinant C-cadherin from
Xenopus
demonstrated that the ectodomain of cadherin exhibits multiple adhesive contacts that involve successive domains along the extracellular region of the protein. Contacts between the fully interdigitated antiparallel proteins form the strongest adhesive interaction. A second weaker minimum was measured when the interdigitated proteins were separated by a distance equal to the length of one domain of the extracellular (EC) fragment and corresponding to the antiparallel alignment of domains one through four (EC1 through EC4). The successive rupture of these interactions generates an unbinding force profile that may be optimized to impede the abrupt failure of cadherin-mediated junctions under force.
Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4D-deficient mice
Authors: Jin S.-L. Catherine , Richard François J. , Kuo Wie-Peng , D’Ercole A. Joseph , Conti Marco
Published: July 2002

In eukaryotic cells, the inactivation of the cyclic nucleotide signal depends on a complex array of cyclic nucleotide phosphodiesterases (PDEs). Although it has been established that multiple PDE isoenzymes with distinct catalytic properties and regulations coexist in the same cell, the physiological significance of this remarkable complexity is poorly understood. To examine the role of a PDE in cAMP signaling
in vivo
, we have inactivated the type 4 cAMP-specific PDE (PDE4D) gene, a mammalian homologue of the
Drosophila
dunce.
This isoenzyme is involved in feedback regulation of cAMP levels. Mice deficient in PDE4D exhibit delayed growth as well as reduced viability and female fertility. The decrease in fertility of the null female is caused by impaired ovulation and diminished sensitivity of the granulosa cells to gonadotropins. These pleiotropic phenotypes demonstrate that PDE4D plays a critical role in cAMP signaling and that the activity of this isoenzyme is required for the regulation of growth and fertility.
Developmental exposure to vasopressin increases aggression in adult prairie voles
Authors: Stribley John M. , Carter C. Sue
Published: July 2002

Although the biological roots of aggression have been the source of intense debate, the precise physiological mechanisms responsible for aggression remain poorly understood. In most species, aggression is more common in males than females; thus, gonadal hormones have been a focal point for research in this field. Although gonadal hormones have been shown to influence the expression of aggression, in many cases aggression can continue after castration, indicating that testicular steroids are not completely essential for the expression of aggression. Recently, the mammalian neuropeptide arginine vasopressin (AVP) has been implicated in aggression. AVP plays a particularly important role in social behavior in monogamous mammals, such as prairie voles (
Microtus ochrogaster
). In turn, the effects of social experiences may be mediated by neuropeptides, including AVP. For example, sexually naïve prairie voles are rarely aggressive. However, 24 h after the onset of mating, males of this species become significantly aggressive toward strangers. Likewise, in adult male prairie voles, central (intracerebroventricular) injections of AVP can significantly increase intermale aggression, suggesting a role for AVP in the expression of postcopulatory aggression in adult male prairie voles. In this paper, we demonstrate that early postnatal exposure to AVP can have long-lasting effects on the tendency to show aggression, producing levels of aggression in sexually naïve, adult male prairie voles that are comparable to those levels observed after mating. Females showed less aggression and were less responsive to exogenous AVP, but the capacity of an AVP V
1a
receptor antagonist to block female aggression also implicates AVP in the development of female aggression.
Crystal structure of
Bacillus subtilis
YabJ, a purine regulatory protein and member of the highly conserved YjgF family
Authors: Sinha Sangita , Rappu Pekka , Lange S. C. , Mäntsälä Pekka , Zalkin Howard , Smith Janet L.
Published: July 2002

The
yabJ
gene in
Bacillus subtilis
is required for adenine-mediated repression of purine biosynthetic genes
in vivo
and codes for an acid-soluble, 14-kDa protein. The molecular mechanism of YabJ is unknown. YabJ is a member of a large, widely distributed family of proteins of unknown biochemical function. The 1.7-Å crystal structure of YabJ reveals a trimeric organization with extensive buried hydrophobic surface and an internal water-filled cavity. The most important finding in the structure is a deep, narrow cleft between subunits lined with nine side chains that are invariant among the 25 most similar homologs. This conserved site is proposed to be a binding or catalytic site for a ligand or substrate that is common to YabJ and other members of the YER057c/YjgF/UK114 family of proteins.
Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events
Authors: Yoon Hye-Joo , Carbon John
Published: July 2002

Yeast two-hybrid and genetic interaction screens indicate that Bir1p, a yeast protein containing phylogenetically conserved antiapoptotic repeat domains called baculovirus inhibitor of apoptosis repeats (BIRs), is involved in chromosome segregation events. In the two-hybrid screen, Bir1p specifically interacts with Ndc10p, an essential component of the yeast kinetochore. Although Bir1p carries two BIR motifs in the N-terminal region, the C-terminal third of the protein is sufficient to provide strong interaction with Ndc10p and moderate interaction with Skp1p, another essential component of the yeast kinetochore. In addition, deletion of
BIR1
is synthetically lethal with deletion of
CBF1
or
CTF19
, genes specifying two other components of the yeast kinetochore. Yeast cells deleted of
BIR1
have a chromosome-loss phenotype, which can be completely rescued by elevating
NDC10
dosage. Furthermore, overexpression of either full-length or the C-terminal region of Bir1p can efficiently suppress the chromosome-loss phenotype of both
bir1
Δ null and
skp1
-
4
mutants. Our data suggest that Bir1p participates in chromosome segregation events, either directly or via interaction with kinetochore proteins, and these effects are apparently not mediated by the BIR domains of Bir1p.
Overexpression of thioredoxin
h
leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain
Authors: Cho Myeong-Je , Wong Joshua H. , Marx Corina , Jiang Wen , Lemaux Peggy G. , Buchanan Bob B.
Published: July 2002

Biochemically active wheat thioredoxin
h
has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin
h
gene (
wtrxh
) were used for transformation; each contained
wtrxh
fused to an endosperm-specific B
1
-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the
bar
herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of
wtrxh
was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin
h
was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin
h
was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B
1
-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin
h
on average than those without a signal sequence. The overexpression of thioredoxin
h
in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves α-1,6 linkages in starch. These results raise the question of how thioredoxin
h
enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.
Structural details of an interaction between cardiolipin and an integral membrane protein
Authors: McAuley Katherine E. , Fyfe Paul K. , Ridge Justin P. , Isaacs Neil W. , Cogdell Richard J. , Jones Michael R.
Published: July 2002

Anionic lipids play a variety of key roles in biomembrane function,
including providing the immediate environment for the integral membrane
proteins that catalyze photosynthetic and respiratory energy
transduction. Little is known about the molecular basis of these
lipid–protein interactions. In this study, x-ray crystallography has
been used to examine the structural details of an interaction between
cardiolipin and the photoreaction center, a key light-driven electron
transfer protein complex found in the cytoplasmic membrane of
photosynthetic bacteria. X-ray diffraction data collected over the
resolution range 30.0–2.1 Å show that binding of the lipid to the
protein involves a combination of ionic interactions between the
protein and the lipid headgroup and van der Waals interactions between
the lipid tails and the electroneutral intramembrane surface of the
protein. In the headgroup region, ionic interactions involve polar
groups of a number of residues, the protein backbone, and bound water
molecules. The lipid tails sit along largely hydrophobic grooves in the
irregular surface of the protein. In addition to providing new
information on the immediate lipid environment of a key integral
membrane protein, this study provides the first, to our knowledge,
high-resolution x-ray crystal structure for cardiolipin. The possible
significance of this interaction between an integral membrane protein
and cardiolipin is considered.
Tonicity-responsive enhancer binding protein, a Rel-like protein that stimulates transcription in response to hypertonicity
Authors: Miyakawa Hiroshi , Woo Seung Kyoon , Dahl Stephen C. , Handler Joseph S. , Kwon H. Moo
Published: July 2002

Hypertonicity (most often present as high salinity) is stressful to the cells of virtually all organisms. Cells survive in a hypertonic environment by increasing the transcription of genes whose products catalyze cellular accumulation of compatible osmolytes. In mammals, the kidney medulla is normally hypertonic because of the urinary concentrating mechanism. Cellular accumulation of compatible osmolytes in the renal medulla is catalyzed by the sodium/
myo
-inositol cotransporter (SMIT), the sodium/chloride/betaine cotransporter, and aldose reductase (synthesis of sorbitol). The importance of compatible osmolytes is underscored by the necrotic injury of the renal medulla and subsequent renal failure that results from the inhibition of SMIT
in vivo
by administration of a specific inhibitor. Tonicity-responsive enhancers (TonE) play a key role in hypertonicity-induced transcriptional stimulation of SMIT, sodium/chloride/betaine cotransporter, and aldose reductase. We report the cDNA cloning of human TonE binding protein (TonEBP), a transcription factor that stimulates transcription through its binding to TonE sequences via a Rel-like DNA binding domain. Western blot and immunohistochemical analyses of cells cultured in hypertonic medium reveal that exposure to hypertonicity elicits slow activation of TonEBP, which is the result of an increase in TonEBP amount and translocation to the nucleus.
Interpreting patterns of gene expression with self-organizing maps: Methods and application to hematopoietic differentiation
Authors: Tamayo Pablo , Slonim Donna , Mesirov Jill , Zhu Qing , Kitareewan Sutisak , Dmitrovsky Ethan , Lander Eric S. , Golub Todd R.
Published: July 2002

Array technologies have made it straightforward to monitor simultaneously the expression pattern of thousands of genes. The challenge now is to interpret such massive data sets. The first step is to extract the fundamental patterns of gene expression inherent in the data. This paper describes the application of self-organizing maps, a type of mathematical cluster analysis that is particularly well suited for recognizing and classifying features in complex, multidimensional data. The method has been implemented in a publicly available computer package,
genecluster
, that performs the analytical calculations and provides easy data visualization. To illustrate the value of such analysis, the approach is applied to hematopoietic differentiation in four well studied models (HL-60, U937, Jurkat, and NB4 cells). Expression patterns of some 6,000 human genes were assayed, and an online database was created.
genecluster
was used to organize the genes into biologically relevant clusters that suggest novel hypotheses about hematopoietic differentiation—for example, highlighting certain genes and pathways involved in “differentiation therapy” used in the treatment of acute promyelocytic leukemia.
Characterization of maize (
Zea mays
L.) Wee1 and its activity in developing endosperm
Authors: Sun Yuejin , Dilkes Brian P. , Zhang Chunsheng , Dante Ricardo A. , Carneiro Newton P. , Lowe Keith S. , Jung Rudolf , Gordon-Kamm William J. , Larkins Brian A.
Published: July 2002

We report the characterization of a maize Wee1 homologue and its expression in developing endosperm. Using a 0.8-kb cDNA from an expressed sequence tag project, we isolated a 1.6-kb cDNA (ZmWee1), which encodes a protein of 403 aa with a calculated molecular size of 45.6 kDa. The deduced amino acid sequence shows 50% identity to the protein kinase domain of human Wee1. Overexpression of ZmWee1 in
Schizosaccharomyces pombe
inhibited cell division and caused the cells to enlarge significantly. Recombinant ZmWee1 obtained from
Escherichia coli
is able to inhibit the activity of p13
suc1
-adsorbed cyclin-dependent kinase from maize. ZmWee1 is encoded by a single gene at a locus on the long arm of chromosome 4. RNA gel blots showed the ZmWee1 transcript is about 2.4 kb in length and that its abundance reaches a maximum 15 days after pollination in endosperm tissue. High levels of expression of ZmWee1 at this stage of endosperm development imply that ZmWee1 plays a role in endoreduplication. Our results show that control of cyclin-dependent kinase activity by Wee1 is conserved among eukaryotes, from fungi to animals and plants.
The role of phosphoglycans inLeishmania–sand fly interactions
Authors: Sacks David L. , Modi Govind , Rowton Edgar , Späth Gerald , Epstein Linda , Turco Salvatore J. , Beverley Stephen M.
Published: July 2002

Leishmaniapromastigotes synthesize an abundance of phosphoglycans, either attached to the cell surface through phosphatidylinositol anchors (lipophosphoglycan, LPG) or secreted as protein-containing glycoconjugates. These phosphoglycans are thought to promote the survival of the parasite within both its vertebrate and invertebrate hosts. The relative contributions of different phosphoglycan-containing molecules inLeishmania–sand fly interactions were tested by using mutants specifically deficient in either total phosphoglycans or LPG alone.Leishmania donovanipromastigotes deficient in both LPG and protein-linked phosphoglycans because of loss ofLPG2(encoding the Golgi GDP-Man transporter) failed to survive the hydrolytic environment within the early blood-fed midgut. In contrast,L. donovaniandLeishmania majormutants deficient solely in LPG expression because of loss ofLPG1(involved in biosynthesis of the core oligosaccharide LPG domain) had only a slight reduction in the survival and growth of promastigotes within the early blood-fed midgut. The ability of theLPG1-deficient promastigotes to persist in the midgut after blood meal excretion was completely lost, and this defect was correlated with their inability to bind to midgut epithelial cellsin vitro. For both mutants, when phosphoglycan expression was restored to wild-type levels by reintroduction ofLPG1orLPG2(as appropriate), then the wild-type phenotype was also restored. We conclude, first, that LPG is not essential for survival in the early blood-fed midgut but, along with other secreted phosphoglycan-containing glycoconjugates, can protect promastigotes from the digestive enzymes in the gut and, second, that LPG is required to mediate midgut attachment and to maintain infection in the fly during excretion of the digested blood meal.
Changes in
period
mRNA levels in the brain and division of labor in honey bee colonies
Authors: Toma Dan P. , Bloch Guy , Moore Darrell , Robinson Gene E.
Published: July 2002

Previous research showed that age-related division of labor in honey bees is associated with changes in activity rhythms; young adult bees perform hive tasks with no daily rhythms, whereas older bees forage with strong daily rhythms. We report that this division of labor is also associated with differences in both circadian rhythms and mRNA levels of
period
, a gene well known for its role in circadian rhythms. The level of
period
mRNA in the brain oscillated in bees of all ages, but was significantly higher at all times in foragers. Elevated
period
mRNA levels cannot be attributed exclusively to aging, because bees induced to forage precociously because of a change in social environment had levels similar to normal age foragers. These results extend the regulation of a “clock gene” to a social context and suggest that there are connections at the molecular level between division of labor and chronobiology in social insects.
Modeling the dynamics of human hair cycles by a follicular automaton
Authors: Halloy J. , Bernard B. A. , Loussouarn G. , Goldbeter A.
Published: July 2002

The hair follicle cycle successively goes through the anagen, catagen, telogen, and latency phases, which correspond, respectively, to hair growth, arrest, shedding, and absence before a new anagen phase is initiated. Experimental observations collected over a period of 14 years in a group of 10 male volunteers, alopecic and nonalopecic, allowed us to determine the characteristics of scalp hair follicle cycles. On the basis of these observations, we propose a follicular automaton model to simulate the dynamics of human hair cycles. The automaton model is defined by a set of rules that govern the stochastic transitions of each follicle between the successive states anagen, telogen, and latency, and the subsequent return to anagen. The transitions occur independently for each follicle, after time intervals given stochastically by a distribution characterized by a mean and a variance. The follicular automaton model accounts both for the dynamical transitions observed in a single follicle and for the behavior of an ensemble of independently cycling follicles. Thus, the model successfully reproduces the evolution of the fractions of follicle populations in each of the three phases, which fluctuate around steady-state or slowly drifting values. We apply the follicular automaton model to the study of spatial patterns of follicular growth that result from a spatially heterogeneous distribution of parameters such as the mean duration of anagen phase. When considering that follicles die or miniaturize after going through a critical number of successive cycles, the model can reproduce the evolution to hair patterns similar to well known types of diffuse or androgenetic alopecia.
Neisseria meningitidisporin PorB interacts with mitochondria and protects cells from apoptosis
Authors: Massari Paola , Ho Yu , Wetzler Lee M.
Published: July 2002

Neisserial porins are strong immune adjuvants and B cell activators. The effect of neisserial porin PorB on activation-induced cell death was investigated, as a potential additional mechanism of the porin's immunopotentiating ability. Neisserial porins interact with target cells to localize intracellularly in the mitochondrial compartment without negatively affecting cellular survival. Pretreatment withNeisseria meningitidisPorB porin decreased or abrogated the mitochondrial damage induced by apoptotic stimuli. In addition, end stage determinants of apoptosis, including DNA breakdown, were diminished by PorB. Immunoprecipitation experiments revealed that PorB interacts with the mitochondrial porin VDAC (voltage-dependent anion channel). The mechanism of the antiapoptotic effect of neisserial porins could be explained by the protein–protein interaction of PorB with VDAC, similar to the interaction of VDAC with antiapoptotic Bcl-2 proteins, resulting in an enhancement of cell survival and continued activation of B cells.
Conditional up-regulation of MHC class I in skeletal muscle leads to self-sustaining autoimmune myositis and myositis-specific autoantibodies
Authors: Nagaraju Kanneboyina , Raben Nina , Loeffler Lisa , Parker Tomasina , Rochon Paul J. , Lee Eunice , Danning Carol , Wada Ryuichi , Thompson Cynthia , Bahtiyar Gul , Craft Joseph , Hooft van Huijsduijnen Rob , Plotz Paul
Published: July 2002

In the human inflammatory myopathies (polymyositis and dermatomyositis), the early, widespread appearance of MHC class I on the surface of muscle cells and the occurrence of certain myositis-specific autoantibodies are striking features. We have used a controllable muscle-specific promoter system to up-regulate MHC class I in the skeletal muscles of young mice. These mice develop clinical, biochemical, histological, and immunological features very similar to human myositis. The disease is inflammatory, limited to skeletal muscles, self-sustaining, more severe in females, and often accompanied by autoantibodies, including, in some mice, autoantibodies to histidyl-tRNA synthetase, the most common specificity found in the spontaneous human disease, anti-Jo-1. This model suggests that an autoimmune disease may unfold in a highly specific pattern as the consequence of an apparently nonspecific event—the sustained up-regulation of MHC class I in a tissue—and that the specificity of the autoantibodies derives not from the specificity of the stimulus, but from the context, location, and probably the duration of the stimulus. This model further suggests that the presumed order of events as an autoimmune disease develops needs to be reconsidered.
Restriction of ectopic recombination by interhomolog interactions during
Saccharomyces cerevisiae
meiosis
Authors: Goldman Alastair S. H. , Lichten Michael
Published: July 2002

In
Saccharomyces cerevisiae
meiosis, recombination occurs frequently between sequences at the same location on homologs (allelic recombination) and can take place between dispersed homologous sequences (ectopic recombination). Ectopic recombination occurs less often than does allelic, especially when homologous sequences are on heterologous chromosomes. To account for this, it has been suggested that homolog pairing (homolog colocalization and alignment) either promotes allelic recombination or restricts ectopic recombination. The latter suggestion was tested by examining ectopic recombination in two cases where normal interhomolog relationships are disrupted. In the first case, one member of a homolog pair was replaced by a homoeologous (related but not identical) chromosome that has diverged sufficiently to prevent allelic recombination. In the second case,
ndj1
mutants were used to delay homolog pairing and synapsis. Both circumstances resulted in a substantial increase in the frequency of ectopic recombination between
arg4
-containing plasmid inserts located on heterologous chromosomes. These findings suggest that, during normal yeast meiosis, progressive homolog colocalization, alignment, synapsis, and allelic recombination restrict the ability of ectopically located sequences to find each other and recombine. In the absence of such restrictions, the meiotic homology search may encompass the entire genome.
Dynamics of dendritic calcium transients evoked by quantal release at excitatory hippocampal synapses
Authors: Murthy Venkatesh N. , Sejnowski Terrence J. , Stevens Charles F.
Published: July 2002

Synaptic
N
-methyl-
d
-aspartate (NMDA) receptors detect coincident pre- and postsynaptic activity and play a critical role in triggering changes in synaptic strength at central synapses. Despite intensive study of synaptic plasticity, relatively little is known about the magnitude and duration of calcium accumulation caused by unitary events at individual synapses. We used fluorescence imaging to detect NMDA receptor-mediated miniature synaptic calcium transients (MSCTs) caused by spontaneous release of synaptic vesicles in dendrites of cultured hippocampal neurons. MSCTs originated focally in dendritic regions <1 μm in length and decayed with a time constant of 0.35 ± 0.03 s. Multiple occurrences of MSCTs recorded at single sites had fluctuating amplitudes, with a coefficient of variation of 0.34. From the reduction in the spatial spread of MSCTs with decreasing concentration of indicator dye, we estimated that the dominant endogenous calcium buffer in dendrites is relatively immobile (diffusion coefficient between 10 and 50 μm
2
/s). We conclude that calcium rise caused by spontaneous quantal synaptic NMDA receptor activation (
i
) is variable, (
ii
) lasts for a time period briefer than previous measurements indicate, and (
iii
) is confined by endogenous calcium buffers to local dendritic regions even when synapses are not on spines.
Rapid induction of senescence in human cervical carcinoma cells
Authors: Goodwin Edward C. , Yang Eva , Lee Chan-Jae , Lee Han-Woong , DiMaio Daniel , Hwang Eun-Seong
Published: July 2002

Expression of the bovine papillomavirus E2 regulatory protein in
human cervical carcinoma cell lines repressed expression of the
resident human papillomavirus E6 and E7 oncogenes and within a few days
caused essentially all of the cells to synchronously display numerous
phenotypic markers characteristic of cells undergoing replicative
senescence. This process was accompanied by marked but in some cases
transient alterations in the expression of cell cycle regulatory
proteins and by decreased telomerase activity. We propose that the
human papillomavirus E6 and E7 proteins actively prevent senescence
from occurring in cervical carcinoma cells, and that once viral
oncogene expression is extinguished, the senescence program is rapidly
executed. Activation of endogenous senescence pathways in
cancer cells may represent an alternative approach to treat human
cancers.
High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia
Authors: Gnaiger Erich , Méndez Gabriela , Hand Steven C.
Published: July 2002

Mitochondria are confronted with low oxygen levels in the
microenvironment within tissues; yet, isolated mitochondria are
routinely studied under air-saturated conditions that are effectively
hyperoxic, increase oxidative stress, and may impair mitochondrial
function. Under hypoxia, on the other hand, respiration and ATP supply
are restricted. Under these conditions of oxygen limitation,
any compromise in the coupling of oxidative phosphorylation to
oxygen consumption could accentuate ATP depletion, leading to
metabolic failure. To address this issue, we have developed the
approach of oxygen-injection microcalorimetry and ADP-injection
respirometry for evaluating mitochondrial function at limiting oxygen
supply. Whereas phosphorylation efficiency drops during ADP limitation
at high oxygen levels, we show here that oxidative phosphorylation is
more efficient at low oxygen than at air saturation, as indicated by
higher ratios of ADP flux to total oxygen flux at identical submaximal
rates of ATP synthesis. At low oxygen, the proton leak and uncoupled
respiration are depressed, thus reducing maintenance energy
expenditure. This indicates the importance of low intracellular oxygen
levels in avoiding oxidative stress and protecting bioenergetic
efficiency.
Identification of a functional transposase of the
Tol2
element, an
Ac
-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage
Authors: Kawakami Koichi , Shima Akihiro , Kawakami Noriko
Published: July 2002

The
Tol2
element of the medaka fish
Oryzias
latipes
belongs to the hAT family of transposons
(
hobo
/
Ac
/
Tam3
). We
report here identification of a functional transposase of
Tol2
that is capable of catalyzing its transposition in
the germ line of zebrafish
Danio rerio
. A transcript
produced from
Tol2
encodes a putative transposase.
Zebrafish fertilized eggs were coinjected with mRNA transcribed
in vitro
, using cDNA of the
Tol2
transcript as a template and a plasmid DNA harboring a mutant
Tol2
, which had a deletion in the putative transposase
gene but retained necessary cis sequences. The injected fish were
raised to adulthood and mated to noninjected fish, and genomic DNA of
the progeny fish were analyzed by PCR and Southern hybridization. Half
of F
1
fish obtained from one of eight injected fish
contained the
Tol2
DNA in their genomes but not the
vector portion. Among these F
1
fish,
Tol2
insertions at four different loci were identified, and some
F
1
fish carried two or three different
Tol2
insertions, indicating that the germ line of the founder fish is highly
mosaic. Sequencing analyses revealed that, in all cases,
Tol2
was surrounded by zebrafish genomic sequences, and
an 8-bp duplication was created at the target site, indicating that
Tol2
was integrated in the zebrafish genome through
transposition. This study identifies an autonomous member of a
DNA-based transposable element from a vertebrate genome. The
Tol2
transposon system should thus be used to develop
novel transgenesis and insertional mutagenesis methods in zebrafish and
possibly in other fishes.
Pho86p, an endoplasmic reticulum (ER) resident protein in
Saccharomyces cerevisiae
, is required for ER exit of the high-affinity phosphate transporter Pho84p
Authors: Lau W.-T. Walter , Howson Russell W. , Malkus Per , Schekman Randy , O'Shea Erin K.
Published: July 2002

In the budding yeast
Saccharomyces cerevisiae
,
PHO84
and
PHO86
are among the genes that are most highly induced in response to phosphate starvation. They are essential for growth when phosphate is limiting, and they function in the high-affinity phosphate uptake system.
PHO84
encodes a high-affinity phosphate transporter, and mutations in
PHO86
cause many of the same phenotypes as mutations in
PHO84
, including a phosphate uptake defect and constitutive expression of the secreted acid phosphatase, Pho5p. Here, we show that the subcellular localization of Pho84p is regulated in response to extracellular phosphate levels; it is localized to the plasma membrane in low-phosphate medium but quickly endocytosed and transported to the vacuole upon addition of phosphate to the medium. Moreover, Pho84p is localized to the endoplasmic reticulum (ER) and fails to be targeted to the plasma membrane in the absence of Pho86p. Utilizing an
in vitro
vesicle budding assay, we demonstrate that Pho86p is required for packaging of Pho84p into COPII vesicles. Pho86p is an ER resident protein, which itself is not transported out of the ER. Interestingly, the requirement of Pho86p for ER exit is specific to Pho84p, because other members of the hexose transporter family to which Pho84 belongs are not mislocalized in the absence of Pho86p.
Circadian modulation of dopamine receptor responsiveness in
Drosophila
melanogaster
Authors: Andretic Rozi , Hirsh Jay
Published: July 2002

We investigated the circadian function of
Drosophila
dopamine receptors by using a behaviorally active decapitated preparation that allows for direct application of drugs to the nerve cord. Quinpirole, a D2-like dopamine receptor agonist, induces reflexive locomotion in decapitated flies. We show that the amount of locomotion induced changes as a function of the time of day, with the highest responsiveness to quinpirole during the subjective night. Furthermore, dopamine receptor responsiveness is under circadian control and depends on the normal function of the
period
gene. The head pacemaker is at least partly dispensable for the circadian modulation of quinpirole-induced locomotion, because changes in agonist responsiveness persist in decapitated flies that are aged for 12 h. This finding suggests a role for the
period
-dependent molecular oscillators in the body in the modulation of amine receptor responsiveness.
Reversal of charge selectivity in transmembrane protein pores by using noncovalent molecular adapters
Authors: Gu Li-Qun , Dalla Serra Mauro , Vincent J. Bryan , Vigh Gyula , Cheley Stephen , Braha Orit , Bayley Hagan
Published: July 2002

In this study, the charge selectivity of staphylococcal α-hemolysin (αHL), a bacterial pore-forming toxin, is manipulated by using cyclodextrins as noncovalent molecular adapters. Anion-selective versions of αHL, including the wild-type pore and various mutants, become more anion selective when β-cyclodextrin (βCD) is lodged within the channel lumen. By contrast, the negatively charged adapter, hepta-6-sulfato-β-cyclodextrin (s
7
βCD), produces cation selectivity. The cyclodextrin adapters have similar effects when placed in cation-selective mutant αHL pores. Most probably, hydrated Cl
−
ions partition into the central cavity of βCD more readily than K
+
ions, whereas s
7
βCD introduces a charged ring near the midpoint of the channel lumen and confers cation selectivity through electrostatic interactions. The molecular adapters generate permeability ratios (P

K
+

/P

Cl
−

) over a 200-fold range and should be useful in the
de novo
design of membrane channels both for basic studies of ion permeation and for applications in biotechnology.
The polycystic kidney disease protein PKD2 interacts with Hax-1, a protein associated with the actin cytoskeleton
Authors: Gallagher Anna Rachel , Cedzich Anna , Gretz Norbert , Somlo Stefan , Witzgall Ralph
Published: July 2002

Despite the recent positional cloning of thePKD1andPKD2genes, which are mutated in the great majority of patients with autosomal-dominant polycystic kidney disease (PKD), the pathogenic mechanism for cyst formation is still unclear. The finding, that the PKD1 and PKD2 proteins interact with each other through their COOH termini, suggests that both proteins are part of the same protein complex or signal transduction pathway. Using a yeast two-hybrid screen with the PKD2 protein, we isolated the PKD2-interacting protein Hax-1. The specificity of the interaction was demonstrated by the fact that PKD2L, a protein closely related to PKD2, failed to interact with Hax-1. Immunofluorescence experiments showed that in most cells PKD2 and Hax-1 colocalized in the cell body, but in some cells PKD2 and Hax-1 also were sorted into cellular processes and lamellipodia. Furthermore we demonstrated an association between Hax-1 and the F-actin-binding protein cortactin, which suggests a link between PKD2 and the actin cytoskeleton. We speculate that PKD2 is involved in the formation of cell-matrix contacts, which are dysfunctional without a wild-type PKD2 protein, thus leading to cystic enlargement of tubular structures in the kidney, liver, and pancreas.
Robust perfect adaptation in bacterial chemotaxis through integral feedback control
Authors: Yi Tau-Mu , Huang Yun , Simon Melvin I. , Doyle John
Published: July 2002

Integral feedback control is a basic engineering strategy for
ensuring that the output of a system robustly tracks its desired value
independent of noise or variations in system parameters. In biological
systems, it is common for the response to an extracellular stimulus to
return to its prestimulus value even in the continued presence of the
signal—a process termed adaptation or desensitization. Barkai, Alon,
Surette, and Leibler have provided both theoretical and experimental
evidence that the precision of adaptation in bacterial chemotaxis is
robust to dramatic changes in the levels and kinetic rate constants of
the constituent proteins in this signaling network [Alon, U., Surette,
M. G., Barkai, N. & Leibler, S. (1998)
Nature
(London)
397, 168–171]. Here we propose that the
robustness of perfect adaptation is the result of this system
possessing the property of integral feedback control. Using techniques
from control and dynamical systems theory, we demonstrate that integral
control is structurally inherent in the Barkai–Leibler model and
identify and characterize the key assumptions of the model. Most
importantly, we argue that integral control in some form is necessary
for a robust implementation of perfect adaptation. More generally,
integral control may underlie the robustness of many homeostatic
mechanisms.
The midblastula transition in
Xenopus
embryos activates multiple pathways to prevent apoptosis in response to DNA damage
Authors: Finkielstein Carla V. , Lewellyn Andrea L. , Maller James L.
Published: July 2002

Apoptosis is controlled by a complex interplay between
regulatory proteins. Previous work has shown that
Xenopus
embryos remove damaged cells by
apoptosis when irradiated before, but not after, the
midblastula transition (MBT). Here we demonstrate that Akt/protein
kinase B is activated and mediates an antiapoptotic signal only
in embryos irradiated after the MBT. In addition, an increase in
xBcl-2/xBax oligomerization and a decrease in xBax homodimerization
promote a protective effect against apoptosis only after the
MBT. The post-MBT survival mechanism arrests cells in G
1
phase by increasing expression of the cyclin-dependent kinase inhibitor
p27
Xic1
. p27
Xic1
associates with cyclin
D/Cdk4 and cyclin A/Cdk2 complexes to cause G
1
/S
arrest, perhaps allowing more time for DNA repair. Taken together, the
results define the DNA damage response as an element of the MBT and
indicate that multiple mechanisms prevent apoptosis after the
MBT.
Mouse model of congenital polycythemia: Homologous replacement of murine gene by mutant human erythropoietin receptor gene
Authors: Divoky Vladimir , Liu Zhiyong , Ryan Thomas M. , Prchal Jaroslav F. , Townes Tim M. , Prchal Josef T.
Published: July 2002

Mutations causing truncations of the cytoplasmic domain of the
human erythropoietin receptor (EPOR) result in a dominantly inherited
disorder—primary familial congenital polycythemia. This disorder is
characterized by increased numbers of erythrocytes (polycythemia) and
by
in vitro
hypersensitivity of erythroid precursors to
erythropoietin. The consequences of EPOR truncation in nonerythroid
tissues are unknown. We replaced the murine EPOR gene with a wild-type
human EPOR gene and a mutant human EPOR gene that we initially
identified in a patient with polycythemia. This mutation leads to an
EPOR truncated after the first tyrosine residue of the intracellular
domain. Mice heterozygous for this mutant allele and a wild-type human
EPOR allele mimicked the human disorder. Interestingly, mice that were
homozygous for the mutant human allele were severely polycythemic but
viable. Our results provide a model for functional studies of
EPOR-triggered signaling pathways in erythropoiesis. These animals can
now be used to investigate the molecular pathophysiology of this
gain-of-function EPOR mutation in erythroid tissue and in those
nonerythroid tissues that express EPOR.