bluuebunny/crossref_metadata_2025 huggingface dataset:
mRNA splicing-mediated C-terminal replacement of transcription factor Hac1p is required for efficient activation of the unfolded protein response
Authors: Mori Kazutoshi , Ogawa Naoki , Kawahara Tetsushi , Yanagi Hideki , Yura Takashi
Published: July 2002

Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR) according to the needs within the ER. In
Saccharomyces cerevisiae
, expression of the UPR-specific transcription factor Hac1p is tightly regulated at the level of mRNA splicing that depends on an unconventional system. Thus,
HAC1
precursor mRNA is constitutively expressed but not translated. A sensor molecule Ire1p/Ern1p-mediated signaling from the ER specifically removes an intron of 252 nucleotides from the precursor mRNA, and the resulting mature mRNA is translated to produce Hac1p. Because the 5′ splice site is located near the C-terminal end of the Hac1p-coding region, this splicing replaces the last 10 codons of the ORF with an exon encoding 18 aa without affecting the N-terminal 220-aa region which contains the DNA-binding domain. Here, we found that this C-terminal 18-aa segment functions as a potent activation domain. Therefore, the splicing event joins the
HAC1
DNA-binding domain to its activation domain, allowing rapid posttranscriptional generation of a potent transcriptional activator (238-aa Hac1p) that activates the UPR efficiently. This suggests that the UPR is hardly activated by Hac1p produced without splicing (230-aa Hac1p) which may occur in the absence of Ire1p/Ern1p-mediated signaling from the ER. Based on these and other results, we propose that the control of expression and activity of Hac1p meets the requirements of the ER.
Generation and rescue of a murine model of platelet dysfunction: The Bernard-Soulier syndrome
Authors: Ware Jerry , Russell Susan , Ruggeri Zaverio M.
Published: July 2002

The human Bernard-Soulier syndrome is an autosomal recessive
disorder of platelet dysfunction presenting with mild thrombocytopenia,
circulating “giant” platelets and a bleeding phenotype. The
bleeding in patients with the Bernard-Soulier syndrome is
disproportionately more severe than suggested by the reduced platelet
count and is explained by a defect in primary hemostasis owing to the
absence of the platelet glycoprotein (GP) Ib-IX-V membrane receptor.
However, the molecular basis for the giant platelet phenotype and
thrombocytopenia have remained unresolved but assumed to be linked to
an absent receptor complex. We have disrupted the gene encoding the
α-subunit of mouse GP Ib-IX-V (GP Ibα) and describe a murine model
recapitulating the hallmark characteristics of the human
Bernard-Soulier syndrome. The results demonstrate a direct link between
expression of a GP Ib-IX-V complex and normal megakaryocytopoiesis and
platelet morphogenesis. Moreover, using transgenic technology the
murine Bernard-Soulier phenotype was rescued by expression of a human
GP Ibα subunit on the surface of circulating mouse platelets. Thus,
an
in vivo
model is defined for analysis of the human GP
Ib-IX-V receptor and its role in the processes performed exclusively by
megakaryocytes and platelets.
Protein factors associated with the SsrA⋅SmpB tagging and ribosome rescue complex
Authors: Karzai A. Wali , Sauer Robert T.
Published: July 2002

SsrA RNA acts as a tRNA and mRNA to modify proteins whose synthesis
on ribosomes has stalled. Such proteins are marked for degradation by
addition of peptide tags to their C termini in a reaction mediated by
SsrA RNA and SmpB, a specific SsrA-RNA binding protein. Evidence is
presented here for the existence of a larger ribonucleoprotein complex
that contains ribosomal protein S1, phosphoribosyl pyrophosphate
synthase, RNase R, and YfbG in addition to SsrA RNA and SmpB.
Biochemical, genetic, and phylogenetic results suggest potential roles
for some of these factors in various stages of the ribosome rescue and
tagging process and/or the presence of functional interactions
between one or more of these proteins and SsrA.
DARPP-32 mediates serotonergic neurotransmission in the forebrain
Authors: Svenningsson Per , Tzavara Eleni T. , Liu Feng , Fienberg Allen A. , Nomikos George G. , Greengard Paul
Published: July 2002

Serotonin is implicated in the regulation of complex sensory, motor, affective, and cognitive functions. However, the biochemical mechanisms whereby this neurotransmitter exerts its actions remain largely unknown. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of molecular weight 32,000) is a phosphoprotein that has primarily been characterized in relation to dopaminergic neurotransmission. Here we report that serotonin regulates DARPP-32 phosphorylation both
in vitro
and
in vivo
. Stimulation of 5-hydroxy-tryptamine (5-HT
4
and 5-HT
6
) receptors causes an increased phosphorylation state at Thr
34
–DARPP-32, the protein kinase A site, and a decreased phosphorylation state at Thr
75
–DARPP-32, the cyclin-dependent kinase 5 site. Furthermore, stimulation of 5-HT
2
receptors increases the phosphorylation state of Ser
137
–DARPP-32, the casein kinase-1 site. Behavioral and gene transcriptional effects induced by compounds that selectively release serotonin were greatly reduced in DARPP-32 knockout mice. Our data indicate that DARPP-32 is essential not only for dopaminergic but also for serotonergic neurotransmission.
Biogeographic range expansion into South America byCoccidioides immitismirrors New World patterns of human migration
Authors: Fisher Matthew C. , Koenig Gina L. , White Thomas J. , San-Blas Gioconda , Negroni Ricardo , Alvarez Isidro Gutiérrez , Wanke Bodo , Taylor John W.
Published: July 2002

Long-distance population dispersal leaves its characteristic signature in genomes, namely, reduced diversity and increased linkage between genetic markers. This signature enables historical patterns of range expansion to be traced. Herein, we use microsatellite loci from the human pathogenCoccidioides immitisto show that genetic diversity in this fungus is geographically partitioned throughout North America. In contrast, analyses of South AmericanC. immitisshow that this population is genetically depauperate and was founded from a single North American population centered in Texas. Variances of allele distributions show that South AmericanC. immitishave undergone rapid population growth, consistent with an epidemic increase in postcolonization population size. Herein, we estimate the introduction into South America to have occurred within the last 9,000–140,000 years. This range increase parallels that ofHomo sapiens. Because of known associations between Amerindians and this fungus, we suggest that the colonization of South America byC. immitisrepresents a relatively recent and rapid codispersal of a host and its pathogen.
A splicing switch and gain-of-function mutation in
FgfR2-IIIc
hemizygotes causes Apert/Pfeiffer-syndrome-like phenotypes
Authors: Hajihosseini Mohammad K. , Wilson Stephen , De Moerlooze Laurence , Dickson Clive
Published: July 2002

Intercellular signaling by fibroblast growth factors plays vital
roles during embryogenesis. Mice deficient for fibroblast growth factor
receptors (FgfRs) show abnormalities in early gastrulation and
implantation, disruptions in epithelial–mesenchymal interactions, as
well as profound defects in membranous and endochondrial bone
formation. Activating
FGFR
mutations are the underlying
cause of several craniosynostoses and dwarfism syndromes in humans.
Here we show that a heterozygotic abrogation of
FgfR2-exon 9
(IIIc)
in mice causes a splicing switch, resulting in a
gain-of-function mutation. The consequences are neonatal growth
retardation and death, coronal synostosis, ocular proptosis, precocious
sternal fusion, and abnormalities in secondary branching in several
organs that undergo branching morphogenesis. This phenotype has strong
parallels to some Apert's and Pfeiffer's syndrome patients.
Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoingin vitrodifferentiation
Authors: Sivori Simona , Falco Michela , Marcenaro Emanuela , Parolini Silvia , Biassoni Roberto , Bottino Cristina , Moretta Lorenzo , Moretta Alessandro
Published: July 2002

In this study we analyzed the progression of cell surface receptor expression during thein vitro-induced human natural killer (NK) cell maturation from CD34+Lin−cell precursors. NKp46 and NKp30, two major triggering receptors that play a central role in natural cytotoxicity, were expressed before the HLA class I-specific inhibitory receptors. Moreover, their appearance at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. Although the early expression of triggering receptors may provide activating signals required for inducing further cell differentiation, it may also affect the self-tolerance of developing NK cells. Our data show that a fail-safe mechanism preventing killing of normal autologous cells may be provided by the 2B4 surface molecule, which, at early stages of NK cell differentiation, functions as an inhibitory rather than as an activating receptor.
Experimental analysis of sperm competition mechanisms in a wild bird population
Authors: Michl Gábor , Török János , Griffith Simon C. , Sheldon Ben C.
Published: July 2002

Recent work on wild birds has revealed the importance of sperm competition as a source of sexual selection, but behavioral and paternity studies have previously provided only indirect evidence for mechanisms of sperm competition in wild birds. In a field study of collared flycatchers
Ficedula albicollis
we used a previously uncharacterized method to determine the frequency and timing of extra-pair inseminations. By counting the number of sperm trapped on the perivitelline layer of eggs, we determined the timing of inseminations and estimated, on a day-to-day basis, the amount of sperm females stored. Our results showed that female collared flycatchers preferentially engaged in extra-pair copulations when mated to an unattractive male with a small white forehead patch. These copulations were timed for the middle part of their fertile period, at least 2 days after the last within-pair insemination. Although the mean number of extra-pair insemination events was only 1.33 per cuckolding female, the ratio between the number of sperm from extra-pair and pair inseminations was at least 5 to 1. Thus a single, well timed extra-pair insemination caused by female behavior could greatly bias fertilization probability in favor of an attractive extra-pair male. Our results suggest a possible behavioral mechanism for female control of sperm competition.
The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability
Authors: Jin Yong Hwan , Obert Robyn , Burgers Peter M. J. , Kunkel Thomas A. , Resnick Michael A. , Gordenin Dmitry A.
Published: July 2002

Many DNA polymerases (Pol) have an intrinsic 3′→5′ exonuclease
(Exo) activity which corrects polymerase errors and prevents mutations.
We describe a role of the 3′→5′ Exo of Pol δ as a supplement or
backup
for the Rad27/Fen1 5′ flap endonuclease. A
yeast
rad27
null allele was lethal in combination with
Pol δ mutations in Exo I, Exo II, and Exo III motifs that inactivate
its exonuclease, but it was viable with mutations in other parts of Pol
δ. The
rad27-p
allele
,
which has little
phenotypic effect by itself, was also lethal in combination with
mutations in the Pol δ Exo I and Exo II motifs. However,
rad27-p
Pol δ Exo III double mutants were viable. They
exhibited strong synergistic increases in
CAN1
duplication mutations, intrachromosomal and interchromosomal
recombination, and required the wild-type double-strand break repair
genes
RAD50
,
RAD51
, and
RAD52
for viability. Observed effects were similar to
those of the
rad27
-null mutant deficient in the removal
of 5′ flaps in the lagging strand. These results suggest that the
3′→5′ Exo activity of Pol δ is redundant with Rad27/Fen1 for
creating ligatable nicks between adjacent Okazaki fragments, possibly
by reducing the amount of strand-displacement in the lagging strand.
Calcium triggers an intramolecular association of the C2 domains in synaptotagmin
Authors: García Ricardo A. , Forde Cameron E. , Godwin Hilary Arnold
Published: July 2002

Synaptotagmin I is a critical component of the synaptic machinery that senses calcium influx and triggers synaptic vesicle fusion and neurotransmitter release. Fluorescence resonance energy transfer studies conducted on synaptotagmin demonstrate that calcium concentrations required for fusion induce a conformational change (EC
50
≈ 3 mM) that brings the two calcium-binding C2 domains in synaptotagmin closer together. Analytical ultracentrifugation studies reveal that synaptotagmin is monomeric under these conditions, indicating that this calcium-triggered association between the C2 domains is intramolecular, rather than intermolecular. These results suggest a mechanism for synaptotagmin function at the presynaptic plasma membrane that involves the self-association of C2 domains.
The ryanodine receptor is essential for larval development in
Drosophila melanogaster
Authors: Sullivan Kathleen M. C. , Scott Kristin , Zuker Charles S. , Rubin Gerald M.
Published: July 2002

We have investigated the role of the ryanodine receptor in
Drosophila
development by using pharmacological and genetic approaches. We identified a
P
element insertion in the
Drosophila
ryanodine receptor gene,
Ryanodine receptor 44F
(
Ryr
), and used it to generate the hypomorphic allele
Ryr

16

. An examination of hypodermal, visceral, and circulatory muscle showed that, in each case, muscle contraction was impaired in
Ryr

16

larvae. Treatment with the drug ryanodine, a highly specific modulator of ryanodine receptor channel activity, also inhibited muscle function, and, at high levels, completely blocked hypodermal muscle contraction. These results suggest that the ryanodine receptor is required for proper muscle function and may be essential for excitation-contraction coupling in larval body wall muscles. Nonmuscle roles of
Ryr
were also investigated. Ryanodine-sensitive Ca
2+
stores had previously been implicated in phototransduction; to address this, we generated
Ryr

16

mutant clones in the adult eye and performed whole-cell, patch–clamp recordings on dissociated ommatidia. Our results do not support a role for
Ryr
in normal light responses.
Induction of cyclooxygenase-2 by Epstein–Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells
Authors: Murono Shigeyuki , Inoue Hiroyasu , Tanabe Tadashi , Joab Irene , Yoshizaki Tomokazu , Furukawa Mitsuru , Pagano Joseph S.
Published: July 2002

Cyclooxygenase-2 (COX-2) is an inducible form of COX and is overexpressed in diverse tumors, raising the possibility of a role for COX-2 in carcinogenesis. In addition, COX-2 contributes to angiogenesis. The Epstein–Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), is detected in at least 70% of nasopharyngeal carcinoma (NPC) and all EBV-infected preinvasive nasopharyngeal lesions. We found that in specimens of LMP1-positive NPC, COX-2 is frequently expressed, whereas LMP1-negative NPC rarely express the enzyme. We next found that expression of LMP1 in EBV-negative nasopharyngeal epithelial cells induced COX-2 expression. Coexpression of IκBα(S32A/S36A), which is not phosphorylated and prevents NF-κB activation, with LMP1 showed that NF-κB is essential for induction of COX-2 by LMP1. We also demonstrate that NF-κB is involved in LMP1-inducedcox-2promoter activity with the use of reporter assays. Two major regions of LMP1, designated CTAR1 and CTAR2, are signal-transducing domains of LMP1. Constructs expressing either CTAR1 or CTAR2 induce COX-2 but to a lesser extent than wild-type LMP1, consistent with the ability of both regions to activate NF-κB. Furthermore, we demonstrate that LMP1-induced COX-2 is functional because LMP1 increased production of prostaglandin E2in a COX-2-dependent manner. Finally, we demonstrate that LMP1 increased production of vascular endothelial growth factor (VEGF). Treatment of LMP1-expressing cells with the COX-2-specific inhibitor (NS-398) dramatically decreased production of VEGF, suggesting that LMP1-induced VEGF production is mediated, at least in part, by COX-2. These results suggest that COX-2 induction by LMP1 may play a role in angiogenesis in NPC.
Kinetic proofreading models for cell signaling predict ways to escape kinetic proofreading
Authors: Hlavacek William S. , Redondo Antonio , Metzger Henry , Wofsy Carla , Goldstein Byron
Published: July 2002

In the context of cell signaling, kinetic proofreading was introduced to explain how cells can discriminate among ligands based on a kinetic parameter, the ligand-receptor dissociation rate constant. In the kinetic proofreading model of cell signaling, responses occur only when a bound receptor undergoes a complete series of modifications. If the ligand dissociates prematurely, the receptor returns to its basal state and signaling is frustrated. We extend the model to deal with systems where aggregation of receptors is essential to signal transduction, and present a version of the model for systems where signaling depends on an extrinsic kinase. We also investigate the kinetics of signaling molecules, “messengers,” that are generated by aggregated receptors but do not remain associated with the receptor complex. We show that the extended model predicts modes of signaling that exhibit kinetic discrimination for some range of parameters but for other parameter values show little or no discrimination and thus escape kinetic proofreading. We compare model predictions with experimental data.
Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome
Authors: Ibrahimi Omar A. , Eliseenkova Anna V. , Plotnikov Alexander N. , Yu Kai , Ornitz David M. , Mohammadi Moosa
Published: July 2002

Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 → Trp and Pro-253 → Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2) . These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2–FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 → Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 → Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients.
Ornaments of the earliest Upper Paleolithic: New insights from the Levant
Authors: Kuhn Steven L. , Stiner Mary C. , Reese David S. , Güleç Erksin
Published: July 2002

Two sites located on the northern Levantine coast, Üçağızlı Cave (Turkey) and Ksar 'Akil (Lebanon) have yielded numerous marine shell beads in association with early Upper Paleolithic stone tools. Accelerator mass spectrometry (AMS) radiocarbon dates indicate ages between 39,000 and 41,000 radiocarbon years (roughly 41,000–43,000 calendar years) for the oldest ornament-bearing levels in Üçağızlı Cave. Based on stratigraphic evidence, the earliest shell beads from Ksar 'Akil may be even older. These artifacts provide some of the earliest evidence for traditions of personal ornament manufacture by Upper Paleolithic humans in western Asia, comparable in age to similar objects from Eastern Europe and Africa. The new data show that the initial appearance of Upper Paleolithic ornament technologies was essentially simultaneous on three continents. The early appearance and proliferation of ornament technologies appears to have been contingent on variable demographic or social conditions.
Stationary-phase mutation in the bacterial chromosome: Recombination protein and DNA polymerase IV dependence
Authors: Bull Harold J. , Lombardo Mary-Jane , Rosenberg Susan M.
Published: July 2002

Several microbial systems have been shown to yield advantageous mutations in slowly growing or nongrowing cultures. In one assay system, the stationary-phase mutation mechanism differs from growth-dependent mutation, demonstrating that the two are different processes. This system assays reversion of a
lac
frameshift allele on an F′ plasmid in
Escherichia coli
. The stationary-phase mutation mechanism at
lac
requires recombination proteins of the RecBCD double-strand-break repair system and the inducible error-prone DNA polymerase IV, and the mutations are mostly −1 deletions in small mononucleotide repeats. This mutation mechanism is proposed to occur by DNA polymerase errors made during replication primed by recombinational double-strand-break repair. It has been suggested that this mechanism is confined to the F plasmid. However, the cells that acquire the adaptive mutations show hypermutation of unrelated chromosomal genes, suggesting that chromosomal sites also might experience recombination protein-dependent stationary-phase mutation. Here we test directly whether the stationary-phase mutations in the bacterial chromosome also occur via a recombination protein- and pol IV-dependent mechanism. We describe an assay for chromosomal mutation in cells carrying the F′
lac
. We show that the chromosomal mutation is recombination protein- and pol IV-dependent and also is associated with general hypermutation. The data indicate that, at least in these male cells, recombination protein-dependent stationary-phase mutation is a mechanism of general inducible genetic change capable of affecting genes in the bacterial chromosome.
In vivoimmunological function of mast cells derived from embryonic stem cells: An approach for the rapid analysis of even embryonic lethal mutations in adult micein vivo
Authors: Tsai Mindy , Wedemeyer Jochen , Ganiatsas Soula , Tam See-Ying , Zon Leonard I. , Galli Stephen J.
Published: July 2002

An important goal of tissue engineering is to achieve reconstitution of specific functionally active cell types by transplantation of differentiated cell populations derived from normal or genetically altered embryonic stem cellsin vitro. We find that mast cells derivedin vitrofrom wild-type or genetically manipulated embryonic stem cells can survive and orchestrate immunologically specific IgE-dependent reactions after transplantation into mast cell-deficientKitW/KitW-vmice. These findings define a unique approach for analyzing the effects of mutations of any genes that are expressed in mast cells, including embryonic lethal mutations,in vitroorin vivo.
Strong antiapostatic selection against novel rare aposematic prey
Authors: Lindström Leena , Alatalo Rauno V. , Lyytinen Anne , Mappes Johanna
Published: July 2002

The evolution of aposematism, a phenomenon where prey species conspicuously advertise their unprofitability to predators, is puzzling. How did conspicuousness evolve, if it simultaneously increased the likelihood of an inexperienced predator to detect the prey and presumably kill it? Antiapostatic selection, where rare prey is predated relatively more often, is considered as another major difficulty for aposematism to evolve. However, the risk of being conspicuous in low frequencies has not been experimentally tested. We designed an experiment to test how frequency (4%, 12%, 32%) of conspicuous aposematic prey and its dispersion type (solitary vs. aggregated) affect an initial predation risk of the prey and in avoidance learning of predators. Wild great tits (
Parus major
) were predators on artificial prey in a “novel world.” The relative mortality of aposematic prey was antiapostatic, thus the frequency-dependent predation was most severe at low frequencies. In all frequencies, aggregated aposematic prey survived better than solitary prey. Surprisingly, learning was not determined by a fixed number of unpalatable prey eaten, but at low frequencies fewer aposematic individuals eaten generated predators' avoidance learning. However, per-capita risk for the prey remained highest at low frequencies. Our results underscore the problems of initial evolution of rare conspicuous morphs. Aggregated prey suffered less from predation, indicating selective advantage of aggregation over solitary living for a conspicuous individual.
The yeast mutant
vps5
Δ affected in the recycling of Golgi membrane proteins displays an enhanced vacuolar Mg
2+
/H
+
exchange activity
Authors: Borrelly Gilles , Boyer Jean-Christophe , Touraine Brigitte , Szponarski Wojciech , Rambier Michèle , Gibrat Rémy
Published: July 2002

Growth of the yeast vacuolar protein-sorting mutant
vps5Δ
affected in the endosome-to-Golgi retromer
complex was more sensitive to Mg
2+
-limiting conditions than
was the growth of the wild-type (WT) strain. This sensitivity was
enhanced at acidic pH. The
vps5Δ
strain was also
sensitive to Al
3+
, known to inhibit Mg
2+
uptake
in yeast cells. In contrast, it was found to be resistant to
Ni
2+
and Co
2+
, two cytotoxic analogs of
Mg
2+
. Resistance to Ni
2+
did not seem to result
from the alteration of plasma-membrane transport properties because
mutant and WT cells displayed similar Ni
2+
uptake. After
plasma-membrane permeabilization, intracellular Ni
2+
uptake
in
vps5Δ
cells was 3-fold higher than in WT cells,
which is consistent with the implication of the vacuole in the observed
phenotypes. In reconstituted vacuolar vesicles prepared from
vps5Δ,
the rates of H
+
exchange with
Ni
2+
, Co
2+
, and Mg
2+
were increased
(relative to WT) by 170%, 130%, and 50%, respectively. The rates of
H
+
exchange with Ca
2+
, Cd
2+
, and
K
+
were similar in both strains, as were α-mannosidase
and H
+
-ATPase activities, and SDS/PAGE patterns of
vacuolar proteins. Among 14 other vacuolar protein-sorting mutants
tested, only the 8 mutants affected in the recycling of trans-Golgi
network membrane proteins shared the same Ni
2+
resistance
phenotype as
vps5Δ.
It is proposed that a trans-Golgi
network Mg
2+
/H
+
exchanger, mislocalized to
vps5Δ
vacuole, could be responsible for the phenotypes
observed
in vivo
and
in vitro.
Mutators and sex in bacteria: Conflict between adaptive strategies
Authors: Tenaillon Olivier , Le Nagard Hervé , Godelle Bernard , Taddei François
Published: July 2002

Bacterial mutation rates can increase and produce genetic novelty, as shown by
in vitro
and
in silico
experiments. Despite the cost due to a heavy deleterious mutation load, mutator alleles, which increase the mutation rate, can spread in asexual populations during adaptation because they remain associated with the rare favorable mutations they generate. This indirect selection for a genetic system generating diversity (second-order selection) is expected to be highly sensitive to changes in the dynamics of adaptation. Here we show by a simulation approach that even rare genetic exchanges, such as bacterial conjugation or transformation, can dramatically reduce the selection of mutators. Moreover, drift or competition between the processes of mutation and recombination in the course of adaptation reveal how second-order selection is unable to optimize the rate of generation of novelty.
Biological clock in total darkness: TheClock/MOP3circadian system of the blind subterranean mole rat
Authors: Avivi Aaron , Albrecht Urs , Oster Henrik , Joel Alma , Beiles Avigdor , Nevo Eviatar
Published: July 2002

Blind subterranean mole rats retain a degenerated, subcutaneous, visually blind but functionally circadian eye involved in photoperiodic perception. Here we describe the cloning, sequence, and expression of the circadianClockandMOP3cDNAs of theSpalax ehrenbergisuperspecies in Israel. Both genes are relatively conserved, although characterized by a significant number of amino acid substitutions. The glutamine-rich area of Clock,which is assumed to function in circadian rhythmicity, is expanded inSpalaxcompared with that of humans and mice, and is different in amino acid composition from that of rats. We also show that MOP3 is a bona fide partner ofSpalaxClock and that theSpalaxClock/MOP3 dimer is less potent than its human counterpart in driving transcription. We suggest that this reduction in transcriptional activity may be attributed to theSpalaxClock glutamine-rich domain, which is unique in its amino acid composition compared with other studied mammalian species. UnderstandingClock/MOP3function could highlight circadian mechanisms in blind mammals and their unique pattern as a result of adapting to life underground.
A physiological role for oxalic acid biosynthesis in the wood-rotting basidiomycete
Fomitopsis palustris
Authors: Munir Erman , Yoon Jeong Jun , Tokimatsu Toshiaki , Hattori Takefumi , Shimada Mikio
Published: July 2002

A metabolic mechanism for oxalic acid biosynthesis in the
wood-rotting basidiomycete
Fomitopsis palustris
has been
proposed on the basis of biochemical analyses of glucose metabolism.
There was a strong correlation between glucose consumption and oxalate
production. Oxalic acid was found to accumulate in the culture fluid in
about 80% of the theoretical yield or about 5-fold, on the basis of
the fungal biomass harvested. The results clearly indicate that glucose
was not completely oxidized to CO
2
by the tricarboxylic
acid (TCA) cycle but converted mainly to oxalate. The determination of
the 12 enzymes concerned has revealed the occurrence of the
unprecedented metabolic coupling of the TCA and glyoxylate cycles that
support oxalate biosynthesis. In this metabolic system, isocitrate
lyase (EC
4.1.3.1
), together with oxaloacetase (EC
3.7.1.1
), was found
to play a pivotal role in yielding oxalate from oxaloacetate via the
acetate-recycling routes. Moreover, malate dehydrogenase (EC
1.1.1.37
),
with an extraordinarily high activity among the enzymes tested, was
shown to play an important role in generating NADH by oxidation of
malate to oxaloacetate. Thus, it is proposed that the wood-rotting
basidiomycete acquires biochemical energy by oxidizing glucose to
oxalate.
Corn pollen deposition on milkweeds in and near cornfields
Authors: Pleasants John M. , Hellmich Richard L. , Dively Galen P. , Sears Mark K. , Stanley-Horn Diane E. , Mattila Heather R. , Foster John E. , Clark Peter , Jones Gretchen D.
Published: July 2002

The density of corn pollen on leaves of milkweed plants inside and outside of cornfields was measured in several studies from different localities. The purpose was to obtain a representative picture of naturally occurring pollen densities to provide a perspective for laboratory and field studies of monarch larvae feeding on milkweed leaves with Bt corn pollen. Pollen density was highest (average 170.6 grains per cm
2
) inside the cornfield and was progressively lower from the field edge outward, falling to 14.2 grains per cm
2
at 2 m. Inside the cornfield, and for each distance from the field edge, a frequency distribution is presented showing the proportion of leaf samples with different pollen densities. Inside cornfields, 95% of leaf samples had pollen densities below 600 grains per cm
2
and the highest pollen density observed was 1400 grains per cm
2
, which occurred in a study with a rainless anthesis period. All other studies had rainfall events during the anthesis period. A single rain event can remove 54–86% of the pollen on leaves. Leaves on the upper portion of milkweed plants, where young monarch larvae tend to feed, had only 30–50% of the pollen density levels of middle leaves.
Phosphorylation near nuclear localization signal regulates nuclear import of adenomatous polyposis coli protein
Authors: Zhang Fang , White Raymond L. , Neufeld Kristi L.
Published: July 2002

Mutation of the adenomatous polyposis coli (
APC
)
gene is an early step in the development of colorectal carcinomas. APC
protein is located in both the cytoplasm and the nucleus. The objective
of this study was to define the nuclear localization signals (NLSs) in
APC protein. APC contains two potential NLSs comprising amino acids
1767–1772 (NLS1
APC
) and 2048–2053 (NLS2
APC
).
Both APC NLSs are well conserved among human, mouse, rat, and fly.
NLS1
APC
and NLS2
APC
each were sufficient to
target the cytoplasmic protein β-galactosidase to the nucleus.
Mutational analysis of APC demonstrated that both NLSs were necessary
for optimal nuclear import of full-length APC protein. Alignment of
NLS2
APC
with the simian virus 40 large T antigen NLS
(NLS
SV40 T-ag
) revealed sequence similarity extending to
adjacent phosphorylation sites. Changing a serine residue
(Ser
2054
) to aspartic acid mutated the potential protein
kinase A site adjacent to NLS2
APC
, resulting in both
inhibition of the NLS2
APC
-mediated nuclear import of a
chimeric β-galactosidase fusion protein and a reduction of
full-length APC nuclear localization. Our data provide evidence that
control of APC's nuclear import through phosphorylation is a potential
mechanism for regulating APC's nuclear activity.
Crystal structure of the precursor of galactose oxidase: An unusual self-processing enzyme
Authors: Firbank S. J. , Rogers M. S. , Wilmot C. M. , Dooley D. M. , Halcrow M. A. , Knowles P. F. , McPherson M. J. , Phillips S. E. V.
Published: July 2002

Galactose oxidase (EC
1.1.3.9
) is a monomeric enzyme that contains a single copper ion and catalyses the stereospecific oxidation of primary alcohols to their corresponding aldehydes. The protein contains an unusual covalent thioether bond between a tyrosine, which acts as a radical center during the two-electron reaction, and a cysteine. The enzyme is produced in a precursor form lacking the thioether bond and also possessing an additional 17-aa pro-sequence at the N terminus. Previous work has shown that the aerobic addition of Cu
2+
to the precursor is sufficient to generate fully processed mature enzyme. The structure of the precursor protein has been determined to 1.4 Å, revealing the location of the pro-sequence and identifying structural differences between the precursor and the mature protein. Structural alignment of the precursor and mature forms of galactose oxidase shows that five regions of main chain and some key residues of the active site differ significantly between the two forms. The precursor structure provides a starting point for modeling the chemistry of thioether bond formation and pro-sequence cleavage.
A formicine in New Jersey Cretaceous amber (Hymenoptera: Formicidae) and early evolution of the ants
Authors: Grimaldi David , Agosti Donat
Published: July 2002

A worker ant preserved with microscopic detail has been discovered in Turonian-aged New Jersey amber [ca.92 mega-annum (Ma)]. The apex of the gaster has an acidopore and, thus, allows definitive assignment of the fossil to the large extant subfamily Formicinae, members of which use a defensive spray of formic acid. This specimen is the only Cretaceous record of the subfamily, and only two other fossil ants are known from the Cretaceous that unequivocally belong to an extant subfamily (BrownimeciaandCanaponeof the Ponerinae, in New Jersey and Canadian amber, respectively). In lieu of a cladogram of formicine genera, generalized morphology of this fossil suggests a basal position in the subfamily. Formicinae and Ponerinae in the mid Cretaceous indicate divergence of basal lineages of ants near the Albian (ca.105–110 Ma) when they presumably diverged from the Sphecomyrminae. Sphecomyrmines are the plesiomorphic sister group to all other ants, or they are a paraphyletic stem group ancestral to all other ants—they apparently became extinct in the Late Cretaceous. Ant abundance in major deposits of Cretaceous and Tertiary insects indicates that they did not become common and presumably dominant in terrestrial ecosystems until the Eocene (ca.45 Ma). It is at this time that modern genera that form very large colonies (at least 10,000 individuals) first appear. During the Cretaceous, eusocial termites, bees, and vespid wasps also first appear—they show a similar pattern of diversification and proliferation in the Tertiary. The Cretaceous ants have further implications for interpreting distributions of modern ants.
Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells
Authors: Sierro Frédéric , Dubois Bertrand , Coste Alix , Kaiserlian Dominique , Kraehenbuhl Jean-Pierre , Sirard Jean-Claude
Published: July 2002

Enteropathogenic bacteria elicit mucosal innate and adaptive immune responses. We investigated whether gut epithelial cells played a role in triggering an adaptive immune response by recruiting dendritic cells (DCs). Immature DCs are selectively attracted by the CCL20 chemokine. The expression of theCCL20gene in human intestinal epithelial cell lines was up-regulated by pathogenic bacteria, includingSalmonellaspecies, but not by indigenous bacteria of the intestinal flora. TheSalmonellamachinery for epithelial cell invasion was not required forCCL20gene activation. Flagellin but not the lipopolysaccharide was found to be theSalmonellafactor responsible for stimulation of epithelial CCL20 production. CCL20 in turn triggered a specific migration of immature DCs. Our data show that crosstalk between bacterial flagellin and epithelial cells is essential for the recruitment of DCs, a mechanism that could be instrumental to initiate adaptive immune responses in the gut.
Support for the multigenic hypothesis of amyloidosis: The binding stoichiometry of retinol-binding protein, vitamin A, and thyroid hormone influences transthyretin amyloidogenicity
in
vitro
Authors: White Joleen T. , Kelly Jeffery W.
Published: July 2002

The amyloidoses are a large group of protein misfolding diseases. Genetic and biochemical evidence support the hypothesis that amyloid formation from wild-type or 1 of 80 sequence variants of transthyretin causes the human amyloid diseases senile systemic amyloidosis or familial amyloid polyneuropathy, respectively. The late onset and variable penetrance of these diseases has led to their designation as multigenic—implying that the expression levels and alleles of multiple gene products influence the course of pathology. Here we show that the binding stoichiometry of three interacting molecules, retinol-binding protein, vitamin A, and
l
-thyroxine, notably influenced transthyretin amyloidogenicity
in vitro
. At least 70 genes control retinol-binding protein, vitamin A, and
l
-thyroxine levels in plasma and have the potential to modulate the course of senile systemic amyloidosis or familial amyloid polyneuropathy.
The calculation of transport properties in quantum liquids using the maximum entropy numerical analytic continuation method: Application to liquid
para
-hydrogen
Authors: Rabani Eran , Reichman David R. , Krilov Goran , Berne Bruce J.
Published: July 2002

We present a method based on augmenting an exact relation between a frequency-dependent diffusion constant and the imaginary time velocity autocorrelation function, combined with the maximum entropy numerical analytic continuation approach to study transport properties in quantum liquids. The method is applied to the case of liquid
para
-hydrogen at two thermodynamic state points: a liquid near the triple point and a high-temperature liquid. Good agreement for the self-diffusion constant and for the real-time velocity autocorrelation function is obtained in comparison to experimental measurements and other theoretical predictions. Improvement of the methodology and future applications are discussed.
Trends of measured climate forcing agents
Authors: Hansen James E. , Sato Makiko
Published: July 2002

The growth rate of climate forcing by measured greenhouse gases
peaked near 1980 at almost 5 W/m
2
per century.
This growth rate has since declined to ≈3 W/m
2
per
century, largely because of cooperative international actions. We argue
that trends can be reduced to the level needed for the moderate
“alternative” climate scenario (≈2 W/m
2
per
century for the next 50 years) by means of concerted actions that have
other benefits, but the forcing reductions are not automatic
“co-benefits” of actions that slow CO
2
emissions.
Current trends of climate forcings by aerosols remain very uncertain.
Nevertheless, practical constraints on changes in emission levels
suggest that global warming at a rate +0.15 ± 0.05°C per decade
will occur over the next several decades.
Ozone-induced responses in Arabidopsis thaliana: the role of salicylic acid in the accumulation of defense-related transcripts and induced resistance.
Authors: Sharma Y K , Léon J , Raskin I , Davis K R
Published: July 2002

Exposure of Arabidopsis thaliana to ozone results in the expression of a number of defense-related genes that are also induced during a hypersensitive response. A potential common link between the activation of defense gene expression during a hypersensitive response and by ozone treatment is the production of active oxygen species and the accumulation of hydrogen peroxide. Here we report that salicylic acid accumulation, which can be induced by hydrogen peroxide and is required for the expression of both a hypersensitive response and systemic acquired resistance, is also required for the induction of some, but not all, ozone-induced mRNAs examined. In addition, we show that ozone exposure triggers induced resistance of A. thaliana to infection with virulent phytopathogenic Pseudomonas syringae strains. Infection of transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of salicylic acid, or npr1 mutant plants, which are defective in the expression of systemic acquired resistance at a step downstream of salicylic acid, demonstrated that the signaling pathway activated during ozone-induced resistance overlaps with the systemic acquired resistance activation pathway and is salicylic acid dependent. Interestingly, plants expressing salicylate hydroxylase exhibited increased sensitivity to ozone exposure. These results demonstrate that ozone activates at least two distinct signaling pathways, including a salicylic acid dependent pathway previously shown to be associated with the activation of pathogen defense reactions, and that this latter pathway also induces a protective response to ozone.
Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae.
Authors: Iyer V , Struhl K
Published: July 2002

We quantitate the absolute levels of individual mRNAs per yeast cell by hybridizing total yeast RNA with an excess of gene-specific 32P-oligonucleotides, and digesting the resulting RNA-DNA hybrids with S1 nuclease. By comparing the his3 hybridization signal from a known amount of yeast cells to the signal generated by a known amount of his3 RNA synthesized in vitro, we determine that yeast strain KY114 growing in yeast extract/peptone/glucose medium at 30 degrees C contains seven molecules of his3 mRNA per cell. Using a galactose shut-off procedure, we determined that the half-life of his3 mRNA is approximately 11 min under these conditions. From these observations, we calculate that one his3 mRNA molecule is synthesized every 140 s. Analysis of other his3 promoter derivatives suggests that the maximal transcriptional initiation rate in yeast cells is one mRNA molecule every 6-8 s. Using his3 as an internal standard, the number of mRNA molecules per cell have been determined for ded1, trp3, rps4, and gall under a variety of growth conditions. From these results, the absolute mRNA level of any yeast gene can be determined in a single hybridization experiment. Moreover, the rate of transcriptional initiation can be determined for mRNAs whose decay rates are known.
Bioactivation of Müllerian inhibiting substance during gonadal development by a kex2/subtilisin-like endoprotease.
Authors: Nachtigal M W , Ingraham H A
Published: July 2002

During male gonadal development Müllerian duct regression is mediated by the actions of the hormone Müllerian inhibiting substance (MIS), a member of the transforming growth factor beta superfamily. MIS is considered to be unique among members of this superfamily because bioactivation of MIS via proteolytic processing is hypothesized to occur at its target organ, the Müllerian duct. We find instead that the majority of MIS is processed and secreted from the embryonic testes as a complex in which the mature region remains noncovalently associated with the prodomain. In addition, we have identified two candidate endoproteases that are expressed in the testes and that may be capable of processing MIS in vivo. These kex2/subtilisin-like enzymes, PC5 and furin, are members of the proprotein convertase family that have been implicated in hormone bioactivation via proteolytic processing after dibasic amino acid cleavage recognition sites. Coexpression of PC5 and MIS in transfected mammalian cells results in efficient processing and bioactivation of MIS. Our results suggest that MIS is a natural substrate for PC5, thereby supporting a role for prohormone convertases in the activation of transforming growth factor beta-related hormones during development.
Active heterodimers are formed from human DNA topoisomerase II alpha and II beta isoforms.
Authors: Biersack H , Jensen S , Gromova I , Nielsen I S , Westergaard O , Andersen A H
Published: July 2002

DNA topoisomerase II is a nuclear enzyme essential for chromosome dynamics and DNA metabolism. In mammalian cells, two genetically and biochemically distinct topoisomerase II forms exist, which are designated topoisomerase II alpha and topoisomerase II beta. In our studies of human topoisomerase II, we have found that a substantial fraction of the enzyme exists as alpha/beta heterodimers in HeLa cells. The ability to form heterodimers was verified when human topoisomerases II alpha and II beta were coexpressed in yeast and investigated in a dimerization assay. Analysis of purified heterodimers shows that these enzymes maintain topoisomerase II specific catalytic activities. The natural existence of an active heterodimeric subclass of topoisomerase II merits attention whenever topoisomerases II alpha and II beta function, localization, and cell cycle regulation are investigated.