bluuebunny/crossref_metadata_2025 huggingface dataset:
Toward a detailed computational model for the mammalian circadian clock
Authors: Leloup Jean-Christophe , Goldbeter Albert
Published: June 2003

We present a computational model for the mammalian circadian clock based on the intertwined positive and negative regulatory loops involving the
Per, Cry, Bmal1, Clock
, and
Rev-Erb
α genes. In agreement with experimental observations, the model can give rise to sustained circadian oscillations in continuous darkness, characterized by an antiphase relationship between
Per
/
Cry
/
Rev-Erb
α and
Bmal1
mRNAs. Sustained oscillations correspond to the rhythms autonomously generated by suprachiasmatic nuclei. For other parameter values, damped oscillations can also be obtained in the model. These oscillations, which transform into sustained oscillations when coupled to a periodic signal, correspond to rhythms produced by peripheral tissues. When incorporating the light-induced expression of the
Per
gene, the model accounts for entrainment of the oscillations by light-dark cycles. Simulations show that the phase of the oscillations can then vary by several hours with relatively minor changes in parameter values. Such a lability of the phase could account for physiological disorders related to circadian rhythms in humans, such as advanced or delayed sleep phase syndrome, whereas the lack of entrainment by light-dark cycles can be related to the non-24h sleep-wake syndrome. The model uncovers the possible existence of multiple sources of oscillatory behavior. Thus, in conditions where the indirect negative autoregulation of
Per
and
Cry
expression is inoperative, the model indicates the possibility that sustained oscillations might still arise from the negative autoregulation of
Bmal1
expression.
Monocyte chemoattractant protein 1 in obesity and insulin resistance
Authors: Sartipy Peter , Loskutoff David J.
Published: June 2003

This study identifies monocyte chemoattractant protein 1 (MCP-1) as an insulin-responsive gene. It also shows that insulin induces substantial expression and secretion of MCP-1 both
in vitro
in insulin-resistant (IR) 3T3-L1 adipocytes and
in vivo
in IR obese mice (
ob
/
ob
). Thus, MCP-1 resembles other previously described genes (e.g.,
PAI-1
and
SREBP-1c
) that remain sensitive to insulin in IR states. The hyperinsulinemia that frequently accompanies obesity and insulin resistance may therefore contribute to the altered expression of these and other genes in insulin target tissues.
In vivo
studies also demonstrate that MCP-1 is overexpressed in obese mice compared with their lean controls, and that white adipose tissue is a major source of MCP-1. The elevated MCP-1 may alter adipocyte function because addition of MCP-1 to differentiated adipocytes
in vitro
decreases insulin-stimulated glucose uptake and the expression of several adipogenic genes (
LpL, adipsin, GLUT-4, aP2
, β3-adrenergic receptor, and peroxisome proliferator-activated receptor γ). These results suggest that elevated MCP-1 may induce adipocyte dedifferentiation and contribute to pathologies associated with hyperinsulinemia and obesity, including type II diabetes.
Liquid–vapor oscillations of water in hydrophobic nanopores
Authors: Beckstein Oliver , Sansom Mark S. P.
Published: June 2003

Water plays a key role in biological membrane transport. In ion channels and water-conducting pores (aquaporins), one-dimensional confinement in conjunction with strong surface effects changes the physical behavior of water. In molecular dynamics simulations of water in short (0.8 nm) hydrophobic pores the water density in the pore fluctuates on a nanosecond time scale. In long simulations (460 ns in total) at pore radii ranging from 0.35 to 1.0 nm we quantify the kinetics of oscillations between a liquid-filled and a vapor-filled pore. This behavior can be explained as capillary evaporation alternating with capillary condensation, driven by pressure fluctuations in the water outside the pore. The free-energy difference between the two states depends linearly on the radius. The free-energy landscape shows how a metastable liquid state gradually develops with increasing radius. For radii > ≈0.55 nm it becomes the globally stable state and the vapor state vanishes. One-dimensional confinement affects the dynamic behavior of the water molecules and increases the self diffusion by a factor of 2–3 compared with bulk water. Permeabilities for the narrow pores are of the same order of magnitude as for biological water pores. Water flow is not continuous but occurs in bursts. Our results suggest that simulations aimed at collective phenomena such as hydrophobic effects may require simulation times >50 ns. For water in confined geometries, it is not possible to extrapolate from bulk or short time behavior to longer time scales.
Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells
Authors: Zalzman Michal , Gupta Sanjeev , Giri Ranjit K. , Berkovich Irina , Sappal Baljit S. , Karnieli Ohad , Zern Mark A. , Fleischer Norman , Efrat Shimon
Published: June 2003

Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.
Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy
Authors: Elenitoba-Johnson Kojo S. J. , Jenson Stephen D. , Abbott Robert T. , Palais Robert A. , Bohling Sandra D. , Lin Zhaosheng , Tripp Sheryl , Shami Paul J. , Wang Lai Y. , Coupland Robert W. , Buckstein Rena , Perez-Ordonez Bayardo , Perkins Sherrie L. , Dube Ian D. , Lim Megan S.
Published: June 2003

Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkin's lymphoma. Transformation to diffuse large B cell lymphoma (DLBCL) is an important cause of mortality. Using cDNA microarray analysis we identified 113 transformation-associated genes whose expression differed consistently between serial clonally related samples of FL and DLBCL occurring within the same individual. Quantitative RT-PCR validated the microarray results and assigned blinded independent group of 20 FLs, 20 DLBCLs, and five transformed lymphoma-derived cell lines with 100%, 70%, and 100% accuracy, respectively. Notably, growth factor cytokine receptors and p38β-mitogen-activated protein kinase (MAPK) were differentially expressed in the DLBCLs. Immunohistochemistry of another blinded set of samples demonstrated expression of phosphorylated p38MAPK in 6/6 DLBCLs and 1/5 FLs, but not in benign germinal centers. SB203580 an inhibitor of p38MAPK specifically induced caspase-3-mediated apoptosis in t(14;18)+/p38MAPK+-transformed FL-derived cell lines. Lymphoma growth was also inhibited in SB203580-treated NOD-SCID mice. Our results implicate p38MAPK dysregulation in FL transformation and suggest that molecular targeting of specific elements within this pathway should be explored for transformed FL therapy.
Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations
Authors: Harley Vincent R. , Layfield Sharon , Mitchell Claire L. , Forwood Jade K. , John Anna P. , Briggs Lyndall J. , McDowall Sharon G. , Jans David A.
Published: June 2003

The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding/bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein
in vitro
and
in vivo
, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMPβ. The SRY-R133W mutant shows reduced IMPβ binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMPβ binding, whereas SRY-R75N and SRY-R76P show normal IMPβ binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.
Myasthenic syndrome caused by mutation of the
SCN4A
sodium channel
Authors: Tsujino Akira , Maertens Chantal , Ohno Kinji , Shen Xin-Ming , Fukuda Taku , Harper C. Michael , Cannon Stephen C. , Engel Andrew G.
Published: June 2003

In a myasthenic syndrome associated with fatigable generalized weakness and recurrent attacks of respiratory and bulbar paralysis since birth, nerve stimulation at physiologic rates rapidly decremented the compound muscle action potential. Intercostal muscle studies revealed no abnormality of the resting membrane potential, evoked quantal release, synaptic potentials, acetylcholine receptor channel kinetics, or endplate ultrastructure, but endplate potentials depolarizing the resting potential to -40 mV failed to excite action potentials. Pursuing this clue, we sequenced
SCN4A
encoding the skeletal muscle sodium channel (Na
v
1.4) and detected two heteroallelic mutations involving conserved residues not present in 400 normal alleles: S246L in the S4/S5 cytoplasmic linker in domain I, and V1442E in the S3/S4 extracellular linker in domain IV. The genetically engineered V1442E-Na channel expressed in HEK cells shows marked enhancement of fast inactivation close to the resting potential, and enhanced use-dependent inactivation on high-frequency stimulation; S246L is likely a benign polymorphism. The V1442E mutation in
SCN4A
defines a novel disease mechanism and a novel phenotype with myasthenic features.
The transient receptor potential channel on the yeast vacuole is mechanosensitive
Authors: Zhou Xin-Liang , Batiza Ann F. , Loukin Stephen H. , Palmer Chris P. , Kung Ching , Saimi Yoshiro
Published: June 2003

Ca
2+
is released from the vacuole into the yeast cytoplasm on an osmotic upshock, but how this upshock is perceived was unknown. We found the vacuolar channel, Yvc1p, to be mechanosensitive, showing that the Ca
2+
conduit is also the sensing molecule. Although fragile, the yeast vacuole allows limited direct mechanical examination. Pressures at tens of millimeters of Hg (1 mmHg = 133 Pa) activate the 400-pS Yvc1p conductance in whole-vacuole recording mode as well as in the excised cytoplasmic-side-out mode. Raising the bath osmolarity activates this channel and causes vacuolar shrinkage and deformation. It appears that, on upshock, a transient osmotic force activates Yvc1p to release Ca
2+
from the vacuole. Mechanical activation of Yvc1p occurs regardless of Ca
2+
concentration and is apparently independent of its known Ca
2+
activation, which we now propose to be an amplification mechanism (Ca
2+
-induced Ca
2+
release). Yvc1p is a member of the transient receptor potential-family channels, several of which have been associated with mechanosensation in animals. The possible use of Yvc1p as a molecular model to study mechanosensation in general is discussed.
A unique mechanism for protein processing and degradation in
Arabidopsis thaliana
Authors: Rojo Enrique , Zouhar Jan , Carter Clay , Kovaleva Valentina , Raikhel Natasha V.
Published: June 2003

Precursor protease vesicles are plant-specific compartments containing precursors of enzymes that are thought to participate in the degradation of cellular components in organs undergoing senescence. We report
in vivo
evidence that the precursor protease vesicle-localized vacuolar processing enzyme-γ (VPEγ) is critical for maturation of the plant vacuolar protease AtCPY. We also provide biochemical and functional evidence that VPEγ is involved in degradation of the vacuolar invertase AtFruct4 in aging tissues. Moreover, a proteomics-based approach identified various proteins found in the vacuoles of aging
vpe
γ mutants but not in WT plants, suggesting a unique role of VPEγ in protein processing and degradation in
Arabidopsis
.
Descent of the larynx in chimpanzee infants
Authors: Nishimura Takeshi , Mikami Akichika , Suzuki Juri , Matsuzawa Tetsuro
Published: June 2003

The human larynx descends during infancy and the early juvenile periods, and this greatly contributes to the morphological foundations of speech development. This developmental phenomenon is believed to be unique to humans. This concept has formed a basis for paleoanthropological studies on the origin and evolution of human speech. We used magnetic resonance imaging to study the development of three living chimpanzees and found that their larynges also descend during infancy, as in human infants. This descent was completed primarily through the rapid descent of the laryngeal skeleton relative to the hyoid, but it was not accompanied by the descent of the hyoid itself. The descent is possibly associated with developmental changes of the swallowing mechanism. Moreover, it contributes physically to an increased independence between the processes of phonation and articulation for vocalization. Thus, the descent of the larynx and the morphological foundations for speech production must have evolved in part during hominoid evolution, and not in a single shift during hominid evolution.
RGS3 mediates a calcium-dependent termination of G protein signaling in sensory neurons
Authors: Tosetti Patrizia , Pathak Narendra , Jacob Michele H. , Dunlap Kathleen
Published: June 2003

G proteins modulate synaptic transmission. Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of Gα subunits, and thus terminate G protein activation. Whether RGS proteins themselves are under cellular control is not well defined, particularly in native cells. In dorsal root ganglion neurons overexpressing RGS3, we find that G protein signaling is rapidly terminated (or “desensitized”) by calcium influx through voltage-gated channels. This rapid desensitization is most likely mediated by direct binding of calcium to RGS3, as deletion of an EF-hand domain in RGS3 abolishes both the desensitization (observed physiologically) and a calcium-RGS3 interaction (observed in a gel-shift assay). A naturally occurring variant of RGS3 that lacks the EF hand neither binds calcium nor produces rapid desensitization, giving rise instead to a slower calcium-dependent desensitization that is attenuated by a calmodulin antagonist. Thus, activity-evoked calcium entry in sensory neurons may provide differential control of G protein signaling, depending on the isoform of RGS3 expressed in the cells. In complex neural circuits subjected to abundant synaptic inhibition by G proteins (as occurs in dorsal spinal cord), rapid termination of inhibition by electrical activity by EF hand-containing RGS3 may ensure the faithful transmission of information from the most active sensory inputs.
Ligand dynamics in a protein internal cavity
Authors: Kriegl Jan M. , Nienhaus Karin , Deng Pengchi , Fuchs Jochen , Nienhaus G. Ulrich
Published: June 2003

We have studied the temperature dependence of the IR stretch bands of carbon monoxide (CO) in the Xe 4 internal cavity of myoglobin mutant L29W-S108L at cryogenic temperatures. Pronounced changes of band areas and positions were analyzed quantitatively by using a simple dynamic model in which CO rotation in the cavity is constrained by a static potential. The librational dynamics of the CO causes a decrease of the total spectral area. A strong local electric field splits the CO stretch absorption into a doublet, indicating that CO can assume opposite orientations in the cavity. With increasing temperature, the two peaks approach each other, because the average angle of the CO with respect to the electric field increases. A combined classical and quantum-mechanical analysis precisely reproduces the observed temperature dependencies of both spectral area and peak shifts. It yields the height of the energy barrier between the two wells associated with opposite CO orientations,V0≈ 2 kJ/mol, and the frequency of oscillation within a well, ω ≈ 25 cm-1. The electric field in the protein cavity was estimated as 10 MV/cm.
Phase transitions and symmetry breaking in singular diffusion
Authors: Denzler Jochen , McCann Robert J.
Published: June 2003

The long-time asymptotics are determined for fast nonlinear diffusion by linearizing Otto's gradient descent model at the Barenblatt profile. The spectrum of the entropy is explicitly determined. The dynamics are found to undergo a phase transition in which rotational symmetry is broken as the strength of the nonlinearity is varied.
UV wavelength-dependent regulation of transcription-coupled nucleotide excision repair in p53-deficient human cells
Authors: Mathonnet Géraldine , Léger Caroline , Desnoyers Julie , Drouin Régen , Therrien Jean-Philippe , Drobetsky Elliot A.
Published: June 2003

Nucleotide excision repair (NER) prevents skin cancer by eliminating highly genotoxic cyclobutane pyrimidine dimers (CPDs) induced in DNA by the UVB component of sunlight. NER consists of two distinct but overlapping subpathways, i.e., global NER, which removes CPD from the genome overall, and transcription-coupled NER (TCNER), which removes CPD uniquely from the transcribed strand of active genes. Previous investigations have clearly established that the p53 tumor suppressor plays a crucial role in the NER process. Here we used the ligation-mediated PCR technique to demonstrate, at nucleotide resolution along two chromosomal genes in human cells, that the requirement for functional p53 in TCNER, but not in global NER, depends on incident UV wavelength. Indeed, relative to an isogenic p53 wild-type counterpart, p53-deficient human lymphoblastoid strains were shown to remove CPD significantly less efficiently along both the transcribed and nontranscribed strands of the
c-jun
and
hprt
loci after exposure to polychromatic UVB (290–320 nm). However, in contrast, after irradiation with 254-nm UV, p53 deficiency engendered less efficient CPD repair only along the nontranscribed strands of these target genes. The revelation of this intriguing wavelength-dependent phenomenon reconciles an apparent conflict between previous studies which used either UVB or 254-nm UV to claim, respectively, that p53 is required for, or plays no role whatsoever in, TCNER of CPD. Furthermore, our finding highlights a major caveat in experimental photobiology by providing a prominent example where the extensively used “nonsolar” model mutagen 254-nm UV does not accurately replicate the effects of environmentally relevant UVB.
Implications of natural selection in shaping 99.4% nonsynonymous DNA identity between humans and chimpanzees: Enlarging genusHomo
Authors: Wildman Derek E. , Uddin Monica , Liu Guozhen , Grossman Lawrence I. , Goodman Morris
Published: June 2003

What do functionally important DNA sites, those scrutinized and shaped by natural selection, tell us about the place of humans in evolution? Here we compare ≈90 kb of coding DNA nucleotide sequence from 97 human genes to their sequenced chimpanzee counterparts and to available sequenced gorilla, orangutan, and Old World monkey counterparts, and, on a more limited basis, to mouse. The nonsynonymous changes (functionally important), like synonymous changes (functionally much less important), show chimpanzees and humans to be most closely related, sharing 99.4% identity at nonsynonymous sites and 98.4% at synonymous sites. On a time scale, the coding DNA divergencies separate the human–chimpanzee clade from the gorilla clade at between 6 and 7 million years ago and place the most recent common ancestor of humans and chimpanzees at between 5 and 6 million years ago. The evolutionary rate of coding DNA in the catarrhine clade (Old World monkey and ape, including human) is much slower than in the lineage to mouse. Among the genes examined, 30 show evidence of positive selection during descent of catarrhines. Nonsynonymous substitutions by themselves, in this subset of positively selected genes, group humans and chimpanzees closest to each other and have chimpanzees diverge about as much from the common human–chimpanzee ancestor as humans do. This functional DNA evidence supports two previously offered taxonomic proposals: family Hominidae should include all extant apes; and genusHomoshould include three extant species and two subgenera,Homo(Homo)sapiens(humankind),Homo(Pan)troglodytes(common chimpanzee), andHomo(Pan)paniscus(bonobo chimpanzee).
Microangiectasias: Structural regulators of lymphocyte transmigration
Authors: Secomb Timothy W. , Konerding Moritz A. , West Charles A. , Su Mei , Young Alan J. , Mentzer Steven J.
Published: June 2003

The migration of lymphocytes into inflammatory tissue requires the migrating cell to overcome mechanical forces produced by blood flow. A generally accepted hypothesis is that these forces are overcome by a multistep sequence of adhesive interactions between lymphocytes and endothelial cells. This hypothesis has been recently challenged by results demonstrating wall shear stress on the order of 20 dyn/cm
2
in vivo
and infrequent lymphocyte–endothelial adhesion at wall shear stress >1–2 dyn/cm
2
in vitro
. Here, we show that lymphocyte slowing and transmigration in the skin is associated with microangiectasias, i.e., focal structural dilatations of microvessel segments. Microangiectasias are inducible within 4 days of the onset of inflammation and lead to a greater than 10-fold local reduction in wall shear stress. These findings support the hypothesis that a preparatory step to lymphocyte transmigration involves structural adaptations in the inflammatory microcirculation.
Breakdown of chlorophyll: A nonenzymatic reaction accounts for the formation of the colorless “nonfluorescent” chlorophyll catabolites
Authors: Oberhuber Michael , Berghold Joachim , Breuker Kathrin , Hörtensteiner Stefan , Kraütler Bernhard
Published: June 2003

Senescent higher plants degrade their chlorophylls (Chls) to polar colorless tetrapyrrolic Chl catabolites, which accumulate in the vacuoles. In extracts from degreened leaves of the tree
Cercidiphyllum japonicum
an unpolar catabolite of this type was discovered. This tetrapyrrole was named
Cj
-NCC-2 and was found to be identical with the product of a stereoselective nonenzymatic isomerization of a “fluorescent” Chl catabolite. This (bio-mimetic) formation of the “nonfluorescent” catabolite
Cj
-NCC-2 took place readily at ambient temperature and at pH 4.9 in aqueous solution. The indicated nonenzymatic process is able to account for a crucial step during Chl breakdown in senescent higher plants. Once delivered to the acidic vacuoles, the fluorescent Chl catabolites are due to undergo a rapid, stereoselective isomerization to the ubiquitous nonfluorescent catabolites. The degradation of the Chl macrocycle is thus indicated to rely on just two known enzymes, one of which is senescence specific and cuts open the chlorin macroring. The two enzymes supply the fluorescent Chl catabolites, which are “programmed” to isomerize further rapidly in an acidic medium, as shown here. Indeed, only small amounts of the latter are temporarily observable during senescence in higher plants.
Pure-tone birdsong by resonance filtering of harmonic overtones
Authors: Beckers Gabriël J. L. , Suthers Roderick A. , Cate Carel ten
Published: June 2003

Pure-tone song is a common and widespread phenomenon in birds. The mechanistic origin of this type of phonation has been the subject of long-standing discussion. Currently, there are three hypotheses. (
i
) A vibrating valve in the avian vocal organ, the syrinx, generates a multifrequency harmonic source sound, which is filtered to a pure tone by a vocal tract filter (“source-filter” model, analogous to human speech production). (
ii
) Vocal tract resonances couple with a vibrating valve source, suppressing the normal production of harmonic overtones at this source (“soprano” model, analogous to human soprano singing). (
iii
) Pure-tone sound is produced as such by a sound-generating mechanism that is fundamentally different from a vibrating valve. Here we present direct evidence of a source-filter mechanism in the production of pure-tone birdsong. Using tracheal thermistors and air sac pressure cannulae, we recorded sound signals close to the syringeal sound source during spontaneous, pure-tone vocalizations of two species of turtledove. The results show that pure-tone dove vocalizations originate through filtering of a multifrequency harmonic sound source.
Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates
Authors: Hardikar Anandwardhan A. , Marcus-Samuels Bernice , Geras-Raaka Elizabeth , Raaka Bruce M. , Gershengorn Marvin C.
Published: June 2003

Development of the endocrine pancreas includes a series of early events wherein precursor cells cluster, that is migrate to form cell aggregates, which subsequently differentiate into islets of Langerhans. We show that PANC-1 cells, a human pancreatic cell line, differentiates into hormone-producing islet-like cell aggregates after exposure to a defined serum-free medium. These cells were used to provide the following evidence that fibroblast growth factor (FGF)2 is a paracrine chemoattractant during PANC-1 cell clustering: (
i
) FGF2 is secreted and remains bound to the extracellular matrix from where it may diffuse to form chemoattractive gradients; (
ii
) a subset of cells expresses FGF receptors (FGFRs) -1, -2, -3, and -4; (
iii
) inhibition of FGFR tyrosine kinase inhibits cell clustering; and (
iv
) FGF2 neutralizing antibody inhibits clustering. In addition, adult human islet-derived precursor cells, which cluster and differentiate in a manner similar to PANC-1 cells, also secrete FGF2 and express FGFRs. We conclude that FGF2, acting as a paracrine chemoattractant, stimulates clustering of precursor cells, an early step leading to islet-like cell aggregate formation. Similar processes may occur during development of the islet of Langerhans in humans.
A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity
Authors: Pfeiffer Julie K. , Kirkegaard Karla
Published: June 2003

Ribavirin is a nucleotide analog that can be incorporated by viral polymerases, causing mutations by allowing base mismatches. It is currently used therapeutically as an antiviral drug during hepatitis C virus infections. During the amplification of poliovirus genomic RNA or hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. This observation has led to the hypothesis that ribavirin's antiviral activity results from error catastrophe caused by increased mutagenesis of viral genomes. Here, we describe the generation of ribavirin-resistant poliovirus by serial viral passage in the presence of increasing concentrations of the drug. Ribavirin resistance can be caused by a single amino acid change, G64S, in the viral polymerase in an unresolved portion of the fingers domain. Compared with wild-type virus, ribavirin-resistant poliovirus displays increased fidelity of RNA synthesis in the absence of ribavirin and increased survival both in the presence of ribavirin and another mutagen, 5-azacytidine. Ribavirin-resistant poliovirus represents an unusual class of viral drug resistance: resistance to a mutagen through increased fidelity.
Large-scale comparison of intron positions in mammalian genes shows intron loss but no gain
Authors: Roy Scott W. , Fedorov Alexei , Gilbert Walter
Published: June 2003

We compared intron–exon structures in 1,560 human–mouse orthologs and 360 mouse–rat orthologs. The origin of differences in intron positions between species was inferred by comparison with an outgroup,
Fugu
for human–mouse and human for mouse–rat. Among 10,020 intron positions in the human–mouse comparison, we found unequivocal evidence for five independent intron losses in the mouse lineage but no evidence for intron loss in humans or for intron gain in either lineage. Among 1,459 positions in rat–mouse comparisons, we found evidence for one loss in rat but neither loss in mouse nor gain in either lineage. In each case, the intron losses were exact, without change in the surrounding coding sequence, and involved introns that are extremely short, with an average of 200 bp, an order of magnitude shorter than the mammalian average. These results favor a model whereby introns are lost through gene conversion with intronless copies of the gene. In addition, the finding of widespread conservation of intron–exon structure, even over large evolutionary distances, suggests that comparative methods employing information about gene structures should be very successful in correctly predicting exon boundaries in genomic sequences.
Predicting gene expression levels from codon biases in α-proteobacterial genomes
Authors: Karlin Samuel , Barnett Melanie J. , Campbell Allan M. , Fisher Robert F. , Mrázek Jan
Published: June 2003

Predicted highly expressed (PHX) genes in five currently available high G+C complete α-proteobacterial genomes are analyzed. These include: the nitrogen-fixing plant symbionts
Sinorhizobium meliloti
(SINME) and
Mesorhizobium loti
(MESLO), the nonpathogenic aquatic bacterium
Caulobacter crescentus
(CAUCR), the plant pathogen
Agrobacterium tumefaciens
(AGRTU), and the mammalian pathogen
Brucella melitensis
(BRUME). Three of these genomes, SINME, AGRTU, and BRUME, contain multiple chromosomes or megaplasmids (>1 Mb length). PHX genes in these genomes are concentrated mainly in the major (largest) chromosome with few PHX genes found in the secondary chromosomes and megaplasmids. Tricarboxylic acid cycle and aerobic respiration genes are strongly PHX in all five genomes, whereas anaerobic pathways of glycolysis and fermentation are mostly not PHX. Only in MESLO (but not SINME) and BRUME are most glycolysis genes PHX. Many flagellar genes are PHX in MESLO and CAUCR, but mostly are not PHX in SINME and AGRTU. The nonmotile BRUME also carries many flagellar genes but these are generally not PHX and all but one are located in the second chromosome. CAUCR stands out among available prokaryotic genomes with 25 PHX TonB-dependent receptors. These are putatively involved in uptake of iron ions and other nonsoluble compounds.
Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry
Authors: Englander Joan J. , Del Mar Charyl , Li Will , Englander S. Walter , Kim Jack S. , Stranz David D. , Hamuro Yoshitomo , Woods Virgil L.
Published: June 2003

An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.
The shape of the Neandertal femur is primarily the consequence of a hyperpolar body form
Authors: Weaver Timothy D.
Published: June 2003

Neandertal femora are distinct from contemporaneous near-modern human femora. Traditionally, these contrasts in femoral shape have been explained as the result of the elevated activity levels and limited cultural abilities of Neandertals. More recently, however, researchers have realized that many of these femoral differences may be explained by the cold-adapted bodies of Neandertals vs. the warm-adapted bodies of near-modern humans. This study explicitly tests this proposed link between climate-induced body proportions and femoral shape by considering the entire hip as a unit by using geometric morphometric methods adapted to deal with articulated structures. Based on recent human patterns of variation, most contrasts in shape between the femora of Neandertals and near-modern humans seem to be secondary consequences of differences in climate-induced body proportions. These results, considered in light of hip mechanics during growth, highlight the importance of developmental and functional integration in determining skeletal form.
Myocardin is a master regulator of smooth muscle gene expression
Authors: Wang Zhigao , Wang Da-Zhi , Pipes G. C. Teg , Olson Eric N.
Published: June 2003

Virtually all smooth muscle genes analyzed to date contain two or more essential binding sites for serum response factor (SRF) in their control regions. Because SRF is expressed in a wide range of cell types, it alone cannot account for smooth muscle-specific gene expression. We show that myocardin, a cardiac muscle- and smooth muscle-specific transcriptional coactivator of SRF, can activate smooth muscle gene expression in a variety of nonmuscle cell types via its association with SRF. Homodimerization of myocardin is required for maximal transcriptional activity and provides a mechanism for cooperative activation of smooth muscle genes by SRF–myocardin complexes bound to different SRF binding sites. These findings identify myocardin as a master regulator of smooth muscle gene expression and explain how SRF conveys smooth muscle specificity to its target genes.
The hSK4 (KCNN4) isoform is the Ca
2
+
-activated K
+
channel (Gardos channel) in human red blood cells
Authors: Hoffman Joseph F. , Joiner William , Nehrke Keith , Potapova Olga , Foye Kristen , Wickrema Amittha
Published: June 2003

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca
2
+
-activated K
+
channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca
2
+
, in the presence but not the absence of external

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and remains refractory to

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added after exposure to inside Ca
2
+
. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside–out patches of red cell membranes, the open probability (
P
o
) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37°C. Net K
+
efflux of intact red cells is also reduced by increasing temperature, as are the
P
o
values of inside–out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.
A role for LIM kinase in cancer invasion
Authors: Yoshioka Kiyoko , Foletta Victoria , Bernard Ora , Itoh Kazuyuki
Published: June 2003

In this study, we show that LIM kinase 1 (LIMK1), a critical regulator of actin dynamics, plays a regulatory role in tumor cell invasion. We found that the level and activity of endogenous LIMK1 is increased in invasive breast and prostate cancer cell lines in comparison with less invasive cells. Overexpression of LIMK1 in MCF-7 and in MDA-MB-231 human breast cancer cell lines increased their motility, whereas the specific ROCK and Rho inhibitors Y-27632 and C3, respectively, attenuated this effect. In addition, inhibition of LIMK1 activity in the MDA-MB-231 cells by expression of dominant-negative LIMK1 resulted in decreased motility and formation of osteolytic bone lesions in an animal model of tumor invasion. This study shows an important role for LIMK1 signaling in invasion of cancer, demonstrating its potential as a therapeutic molecular target to decrease metastasis.
Behavioral performance in adult honey bees is influenced by the temperature experienced during their pupal development
Authors: Tautz Jürgen , Maier Sven , Groh Claudia , Rössler Wolfgang , Brockmann Axel
Published: June 2003

To investigate the possible consequences of brood-temperature regulation in honey bee colonies on the quality of behavioral performance of adults, we placed honey bee pupae in incubators and allowed them to develop at temperatures held constant at 32°C, 34.5°C, and 36°C. This temperature range occurs naturally within hives. On emergence, the young adult bees were marked and introduced into foster colonies housed in normal and observation hives and allowed to live out their lives. No obvious difference in within-hive behavior was noted between the temperature-treated bees and the foster-colony bees. However, when the temperature-treated bees became foragers and were trained to visit a feeder 200 m from the hive, they exhibited clear differences in dance performance that could be correlated with the temperatures at which they had been raised: bees raised at 32°C completed only ≈20% of the dance circuits when compared with bees of the higher-temperature group. Also, the variance in the duration of the waggle phase is larger in 32°C-raised bees compared with 36°C-raised bees. All other parameters compared across all groups were not significantly different. One-trial learning and memory consolidation in the bees raised at different temperatures was investigated 1 and 10 min after conditioning the proboscis-extension reflex. Bees raised at 36°C performed as expected for bees typically classified as “good learners,” whereas bees raised at 32°C and 34.5°C performed significantly less well. We propose that the temperature at which pupae are raised will influence their behavioral performance as adults and may determine the tasks they carry out best inside and outside the hive.
An autoregulatory loop controls peroxisome proliferator-activated receptor γ coactivator 1α expression in muscle
Authors: Handschin Christoph , Rhee James , Lin Jiandie , Tarr Paul T. , Spiegelman Bruce M.
Published: June 2003

Skeletal muscle adapts to chronic physical activity by inducing mitochondrial biogenesis and switching proportions of muscle fibers from type II to type I. Several major factors involved in this process have been identified, such as the calcium/calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A (CnA), and the transcriptional component peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Transgenic expression of PGC-1α recently has been shown to dramatically increase the content of type I muscle fibers in skeletal muscle, but the relationship between PGC-1α expression and the key components in calcium signaling is not clear. In this report, we show that the PGC-1α promoter is regulated by both CaMKIV and CnA activity. CaMKIV activates PGC-1α largely through the binding of cAMP response element-binding protein to the PGC-1α promoter. Moreover, we show that a positive feedback loop exists between PGC-1α and members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. MEF2s bind to the PGC-1α promoter and activate it, predominantly when coactivated by PGC-1α. MEF2 activity is stimulated further by CnA signaling. These findings imply a unified pathway, integrating key regulators of calcium signaling with the transcriptional switch PGC-1α. Furthermore, these data suggest an autofeedback loop whereby the calcium-signaling pathway may result in a stable induction of PGC-1α, contributing to the relatively stable nature of muscle fiber-type determination.
Variation in
Ginkgo biloba
L. leaf characters across a climatic gradient in China
Authors: Sun Bainian , Dilcher David L. , Beerling David J. , Zhang Chengjun , Yan Defei , Kowalski Elizabeth
Published: June 2003

Fossil leaves assigned to the genus
Ginkgo
are increasingly being used to reconstruct Mesozoic and Tertiary environments based on their stomatal and carbon isotopic characteristics. We sought to provide a more secure basis for understanding variations seen in the plant fossil record by determining the natural variability of these properties of sun and shade leaf morphotypes of
Ginkgo biloba
trees under the present atmospheric CO
2
concentration and a range of contemporary climates in three Chinese locations (Lanzhou, Beijing, and Nanjing). Climate had no major effects on leaf stomatal index (proportion of leaf surface cells that are stomata) but did result in more variable stomatal densities. The effects of climate and leaf morphotype on stomatal index were rather conserved (<1%) and much less than the response of trees to recent CO
2
increases. Leaf carbon isotope discrimination (Δ) was highest for trees in Nanjing, which experience a warm, moist climate, whereas trees in the most arid site (Lanzhou) had the lowest Δ values. Interestingly, the variation in Δ shown by leaf populations of trees from China and the United Kingdom was very similar to that of fossil
Ginkgo
cuticles dating to the Mesozoic and Tertiary, which suggests to us that the physiology of leaf carbon uptake and regulation of water loss in
Ginkgo
has remained highly conserved despite the potential for evolutionary change over millions of years.
Protection and compensation in the influenza virus-specific CD8+T cell response
Authors: Webby Richard J. , Andreansky Samita , Stambas John , Rehg Jerold E. , Webster Robert G. , Doherty Peter C. , Turner Stephen J.
Published: June 2003

Influenza virus-specific CD8+T cells generally recognize peptides derived from conserved, internal proteins that are not subject to antibody-mediated selection pressure. Prior exposure to any one influenza A virus (H1N1) can prime for a secondary CD8+T cell response to a serologically different influenza A virus (H3N2). The protection afforded by this recall of established CD8+T cell memory, although limited, is not negligible. Key characteristics of primary and secondary influenza-specific host responses are probed here with recombinant viruses expressing modified nucleoprotein (NP) and acid polymerase (PA) genes. Point mutations were introduced into the epitopes derived from the NP and PA such that they no longer bound the presenting H2DbMHC class I glycoprotein, and reassortant H1N1 and H3N2 viruses were made by reverse genetics. Conventional (C57BL/6J, H2b, and Ig+/+) and Ig-/-(μMT) mice were more susceptible to challenge with the single NP [HKx31 influenza A virus (HK)-NP] and PA (HK-PA) mutants, but unlike the Ig-/-mice, Ig+/+mice were surprisingly resistant to the HK-NP/-PA double mutant. This virus was found to promote an enhanced IgG response resulting, perhaps, from the delayed elimination of antigen-presenting cells. Antigen persistence also could explain the increase in size of the minor KbPB1703CD8+T cell population in mice infected with the mutant viruses. The extent of such compensation was always partial, giving the impression that any virus-specific CD8+T cell response operates within constrained limits. It seems that the relationship between protective humoral and cellular immunity is neither simple nor readily predicted.
Rapid biosynthesis of
N
-linolenoyl-
l
-glutamine, an elicitor of plant volatiles, by membrane-associated enzyme(s) in
Manduca sexta
Authors: Lait Cameron G. , Alborn Hans T. , Teal Peter E. A. , Tumlinson James H.
Published: June 2003

In response to elicitors in the oral secretions of caterpillars, plants produce and release volatile chemicals that attract predators and parasitoids of the caterpillar while it feeds. The most prevalent elicitors are fatty acid amides consisting of 18-carbon polyunsaturated fatty acids coupled with
l
-glutamine. We demonstrate rapid CoA- and ATP-independent
in vitro
biosynthesis of the fatty acid amide elicitor,
N
-linolenoyl-
l
-glutamine, by microsomal fractions of several alimentary tissues in
Manduca sexta. N
-linolenoyl-
l
-glutamine is a structural analog of several other elicitors including volicitin, the first fatty acid amide elicitor identified in caterpillars. The enzyme(s) that catalyzed biosynthesis of
N
-linolenoyl-
l
-glutamine was localized within the integral membrane protein fraction extracted from microsomes by Triton X-114 detergent phase partitioning and had maximum activity at alkaline pH. We found no evidence suggesting microbial or tissue-independent biosynthesis of
N
-linolenoyl-
l
-glutamine in
M. sexta
. The
in vitro
biosynthesis of
N
-linolenoyl-
l
-glutamine by membrane-associated enzyme(s) in
M. sexta
represents direct evidence of fatty acid amide synthesis by caterpillar tissues.