bluuebunny/crossref_metadata_2025 huggingface dataset:
Removal of polymerase-produced mutant sequences from PCR products
Authors: Smith Jane , Modrich Paul
Published: July 2002

Heteroduplex DNA lacking d(GATC) methylation is subject to mismatch-provoked double-strand cleavage at d(GATC) sites in a reaction dependent on MutH, MutL, MutS, and ATP. We have exploited this reaction to develop a method for removal of polymerase-produced mutant sequences that arise during sequence amplification by PCR. After denaturation and reannealing, the PCR product pool is subjected to MutH, MutL, and MutS mismatch repair proteins under double-strand cleavage conditions, followed by isolation of uncleaved product by size selection. Use of an
Escherichia coli lac
forward mutation assay has shown that this procedure reduces the incidence of polymerase-induced mutant sequences by an order of magnitude. Twenty mutants that originated from three independent PCR amplification reactions and survived MutHLS treatment all were found to contain an infrequently occurring A⋅T → T⋅A transversion mutation at a unique position within the product. By contrast, the majority of mutations in untreated PCR products were transitions occurring throughout the amplified region, although frameshifts and transversions also were observed. The MutHLS method thus can be used to effectively remove the majority of mutant sequences produced by polymerase errors during PCR amplification.
Voltage dependence of the pattern and frequency of discrete Ca
2+
release events after brief repriming in frog skeletal muscle
Authors: Klein Michael G. , Lacampagne Alain , Schneider Martin F.
Published: July 2002

Applying a brief repolarizing pre-pulse to a depolarized frog skeletal muscle fiber restores a small fraction of the transverse tubule membrane voltage sensors from the inactivated state. During a subsequent depolarizing test pulse we detected brief, highly localized elevations of myoplasmic Ca
2+
concentration (Ca
2+
“sparks”) initiated by restored voltage sensors in individual triads at all test pulse voltages. The latency histogram of these events gives the gating pattern of the sarcoplasmic reticulum (SR) calcium release channels controlled by the restored voltage sensors. Both event frequency and clustering of events near the start of the test pulse increase with test pulse depolarization. The macroscopic SR calcium release waveform, obtained from the spark latency histogram and the estimated open time of the channel or channels underlying a spark, exhibits an early peak and rapid marked decline during large depolarizations. For smaller depolarizations, the release waveform exhibits a smaller peak and a slower decline. However, the mean use time and mean amplitude of the individual sparks are quite similar at all test depolarizations and at all times during a given depolarization, indicating that the channel open times and conductances underlying sparks are essentially independent of voltage. Thus, the voltage dependence of SR Ca
2+
release is due to changes in the frequency and pattern of occurrence of individual, voltage-independent, discrete release events.
Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug
Authors: Fuentes-Prior Pablo , Noeske-Jungblut Christiane , Donner Peter , Schleuning Wolf-Dieter , Huber Robert , Bode Wolfram
Published: July 2002

Triabin, a 142-residue protein from the saliva of the blood-sucking triatomine bug
Triatoma pallidipennis
, is a potent and selective thrombin inhibitor. Its stoichiometric complex with bovine α-thrombin was crystallized, and its crystal structure was solved by Patterson search methods and refined at 2.6-Å resolution to an
R
value of 0.184. The analysis revealed that triabin is a compact one-domain molecule essentially consisting of an eight-stranded β-barrel. The eight strands A to H are arranged in the order A-C-B-D-E-F-G-H, with the first four strands exhibiting a hitherto unobserved up-up-down-down topology. Except for the B-C inversion, the triabin fold exhibits the regular up-and-down topology of lipocalins. In contrast to the typical ligand-binding lipocalins, however, the triabin barrel encloses a hydrophobic core intersected by a unique salt-bridge cluster. Triabin interacts with thrombin exclusively via its fibrinogen-recognition exosite. Surprisingly, most of the interface interactions are hydrophobic. A prominent exception represents thrombin’s Arg-77A side chain, which extends into a hydrophobic triabin pocket forming partially buried salt bridges with Glu-128 and Asp-135 of the inhibitor. The fully accessible active site of thrombin in this complex is in agreement with its retained hydrolytic activity toward small chromogenic substrates. Impairment of thrombin’s fibrinogen converting activity or of its thrombomodulin-mediated protein C activation capacity upon triabin binding is explained by usage of overlapping interaction sites of fibrinogen, thrombomodulin, and triabin on thrombin. These data demonstrate that triabin inhibits thrombin via a novel and unique mechanism that might be of interest in the context of potential therapeutic applications.
Dependence of intracellular signaling and neurosecretion on phospholipase D activation in immortalized gonadotropin-releasing hormone neurons
Authors: Zheng Lixin , Krsmanovic Lazar Z. , Vergara Leoncio A. , Catt Kevin J. , Stojilkovic Stanko S.
Published: July 2002

The excitability of gonadotropin-releasing hormone (GnRH) neurons is essential for episodic neuropeptide release, but the mechanism by which electrical activity controls GnRH secretion is not well characterized. The role of phospholipase D (PLD) in mediating the activity-dependent secretory pathway was investigated in immortalized GT1 neurons, which both secrete GnRH and express GnRH receptors. Activation of these Ca
2+
-mobilizing receptors was associated with transient hyperpolarization of GT1 cells, followed by sustained firing of action potentials. This was accompanied by an increase in PLD activity, as indicated by elevated phosphatidylethanol (PEt) production. GnRH-induced PEt production was reduced by inhibition of phospholipase C-dependent phosphoinositide hydrolysis by U73122 and neomycin, suggesting that signaling from phospholipase C led to activation of PLD. The intermediate role of protein kinase C (PKC) in this process was indicated by the ability of phorbol 12-myristate 13-acetate to induce time- and dose-dependent increases in PEt and diacylglycerol, but not inositol trisphosphate, and by reduction of GnRH-induced PEt accumulation in PKC-depleted cells. Consistent with the role of action potential-driven Ca
2+
entry in this process, agonist-induced PLD activity was also reduced by nifedipine and low extracellular Ca
2+
. Inhibition of the PLD pathway by ethanol and propranolol reduced diacylglycerol production and caused a concomitant fall in GnRH release. These data indicate that voltage-gated Ca
2+
entry and PKC act in an independent but cooperative manner to regulate PLD activity, which contributes to the secretory response in GT1 cells. Thus, the electrical activity of the GnRH-secreting neuron participates in the functional coupling between GnRH receptors and PLD pathway.
Ensemble quantum computing by NMR spectroscopy
Authors: Cory David G. , Fahmy Amr F. , Havel Timothy F.
Published: July 2002

A quantum computer (QC) can operate in parallel on all its possible
inputs at once, but the amount of information that can be extracted
from the result is limited by the phenomenon of wave function collapse.
We present a new computational model, which differs from a QC only in
that the result of a measurement is the expectation value of the
observable, rather than a random eigenvalue thereof. Such an
expectation value QC can solve nondeterministic polynomial-time
complete problems in polynomial time. This observation is significant
precisely because the computational model can be realized, to a certain
extent, by NMR spectroscopy on macroscopic ensembles of quantum spins,
namely molecules in a test tube. This is made possible by identifying a
manifold of statistical spin states, called pseudo-pure states, the
mathematical description of which is isomorphic to that of an isolated
spin system. The result is a novel NMR computer that can be programmed
much like a QC, but in other respects more closely resembles a DNA
computer. Most notably, when applied to intractable combinatorial
problems, an NMR computer can use an amount of sample, rather than
time, which grows exponentially with the size of the problem. Although
NMR computers will be limited by current technology to exhaustive
searches over only 15 to 20 bits, searches over as much as 50 bits are
in principle possible, and more advanced algorithms could greatly
extend the range of applicability of such machines.
Functional organization of spatial and nonspatial working memory processing within the human lateral frontal cortex
Authors: Owen Adrian M. , Stern Chantal E. , Look Rodney B. , Tracey Irene , Rosen Bruce R. , Petrides Michael
Published: July 2002

The present study used functional magnetic resonance imaging to demonstrate that performance of visual spatial and visual nonspatial working memory tasks involve the same regions of the lateral prefrontal cortex when all factors unrelated to the type of stimulus material are appropriately controlled. These results provide evidence that spatial and nonspatial working memory may not be mediated, respectively, by mid-dorsolateral and mid-ventrolateral regions of the frontal lobe, as widely assumed, and support the alternative notion that specific regions of the lateral prefrontal cortex make identical executive functional contributions to both spatial and nonspatial working memory.
Local mechanical stimulation induces components of the pathogen defense response in parsley
Authors: Gus-Mayer Sabine , Naton Beatrix , Hahlbrock Klaus , Schmelzer Elmon
Published: July 2002

Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response toPhytophthora sojae. In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli. Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes. When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected. A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation. By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations. Neither papilla formation nor hypersensitive cell death occurred under the conditions tested. These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes. As yet unidentified signals may be involved in papilla formation and hypersensitive cell death.
Determining and dating recent rodent speciation events by using L1 (LINE-1) retrotransposons
Authors: Verneau Olivier , Catzeflis François , Furano Anthony V.
Published: July 2002

Phylogenies based on the inheritance of shared derived characters will be ambiguous when the shared characters are not the result of common ancestry. Such characters are called homoplasies. Phylogenetic analysis also can be problematic if the characters have not changed sufficiently, as might be the case for rapid or recent speciations. The latter are of particular interest because evolutionary processes may be more accessible the more recent the speciation. The repeated DNA subfamilies generated by the mammalian L1 (LINE-1) retrotransposon are apparently homoplasy-free phylogenetic characters. L1 retrotransposons are transmitted only by inheritance and rapidly generate novel variants that produce distinct subfamilies of mostly defective copies, which then “age” as they diverge. Here we show that the L1 character can both resolve and date recent speciation events within the large group of very closely related rats known as
Rattus
sensu stricto. This lineage arose 5–6 million years ago (Mya) and subsequently underwent two episodes of speciation: an intense one, ≈2.7 Mya, produced at least five lineages in <0.3 My; a second began ≈1.2 Mya and may still be continuing.
A dysfunctional desmin mutation in a patient with severe generalized myopathy
Authors: Muñoz-Mármol Ana M. , Strasser Geraldine , Isamat Marcos , Coulombe Pierre A. , Yang Yanmin , Roca Xavier , Vela Elena , Mate José L. , Coll Jaume , Fernández-Figueras María Teresa , Navas-Palacios José J. , Ariza Aurelio , Fuchs Elaine
Published: July 2002

Mice lacking desmin produce muscle fibers with Z disks and normal sarcomeric organization. However, the muscles are mechanically fragile and degenerate upon repeated contractions. We report here a human patient with severe generalized myopathy and aberrant intrasarcoplasmic accumulation of desmin intermediate filaments. Muscle tissue from this patient lacks the wild-type desmin allele and has a desmin gene mutation encoding a 7-aa deletion within the coiled-coil segment of the protein. We show that recombinant desmin harboring this deletion cannot form proper desmin intermediate filament networks in cultured cells, nor is it able to assemble into 10-nm filaments
in vitro
. These findings provide direct evidence that a mutation in desmin can cause human myopathies.
The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein
Authors: Puri Anu , Hug Peter , Jernigan Kristine , Barchi Joseph , Kim Hee-Yong , Hamilton Jillon , Wiels Joëlle , Murray Gary J. , Brady Roscoe O. , Blumenthal Robert
Published: July 2002

Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4+or GSL-depleted human CD4+cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4+cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1LAIenvelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4+nonhuman and GSL-depleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(α1→4)Gal(β1→4)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or α-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4/CXCR4-dependent HIV-1 fusion.
Specific requirement for CD3ɛ in T cell development
Authors: DeJarnette Jan B. , Sommers Connie L. , Huang Kun , Woodside Kenneth J. , Emmons Rebecca , Katz Kenneth , Shores Elizabeth W. , Love Paul E.
Published: July 2002

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3γ, -δ, -ɛ, and ζ). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3γ, -δ, or ζ results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3ɛ
−/−
mice, and thymocyte development is arrested at the early CD4
−
CD8
−
stage. Although these results suggest that CD3ɛ is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3γ and CD3δ genes also is reduced in CD3ɛ
−/−
mice. Thus, it is unclear whether the phenotype of CD3ɛ
−/−
mice reflects the collective effects of CD3γ, -δ, and -ɛ deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3ɛ gene via Cre/
lox
P-mediated recombination, we generated mice that lack CD3ɛ yet retain normal expression of the closely linked CD3γ and CD3δ genes. These (CD3ɛ
Δ/Δ
) mice exhibited an early arrest in T cell development, similar to that of CD3ɛ
−/−
mice. Moreover, the developmental defect could be rescued by expression of a CD3ɛ transgene. These results identify an essential role for CD3ɛ in T cell development not shared by the CD3γ, CD3δ, or ζ-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity.
Reduced growth, abnormal kidney structure, and type 2 (AT
2
) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT
1A
and AT
1B
receptors for angiotensin II
Authors: Oliverio Michael I. , Kim Hyung-Suk , Ito Masaki , Le Thu , Audoly Laurent , Best Christopher F. , Hiller Sylvia , Kluckman Kimberly , Maeda Nobuyo , Smithies Oliver , Coffman Thomas M.
Published: July 2002

The classically recognized functions of the renin–angiotensin system are mediated by type 1 (AT
1
) angiotensin receptors. Whereas man possesses a single AT
1
receptor, there are two AT
1
receptor isoforms in rodents (AT
1A
and AT
1B
) that are products of separate genes (
Agtr1a
and
Agtr1b
). We have generated mice lacking AT
1B
(
Agtr1b
−/−) and both AT
1A
and AT
1B
receptors (
Agtr1a
−/−
Agtr1b
−/−).
Agtr1b
−/− mice are healthy, without an abnormal phenotype. In contrast,
Agtr1a
−/−
Agtr1b
−/− mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (
Agt
−/−) and angiotensin-converting enzyme-deficient (
Ace
−/−) mice that are unable to synthesize angiotensin II.
Agtr1a
−/−
Agtr1b
−/− mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the
Agtr1a
−/−
Agtr1b
−/− mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT
2
-receptor signaling. In summary, our studies suggest that both AT
1
receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT
1
receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT
1
receptors to mediate physiological functions of the renin–angiotensin system.
Cloning and functional expression of alternative spliced variants of the ρ1 γ-aminobutyrate receptor
Authors: Martínez-Torres Ataúlfo , Vazquez Ana E. , Panicker Mitradas M. , Miledi Ricardo
Published: July 2002

The ρ1 γ-aminobutyrate receptor (GABA
ρ1
) is expressed predominantly in the retina and forms homomeric GABA-gated Cl
−
channels that are clearly different from the multisubunit GABA
A
receptors. In contrast to these, GABA
ρ1
receptors desensitize very little and are not blocked by bicuculline. In addition to GABA
ρ1
, two new variants were identified in human retina cDNA libraries. Cloning and sequence analysis showed that both variants contain large deletions in the putative extracellular domain of the receptor. These deletions extend from a common 5′ site to different 3′ sites. The cDNA with the largest deletion, named GABA
ρ1
Δ450, contains a complete ORF identical to that of GABA
ρ1
but missing 450 nt. This cDNA encodes a protein of 323 aa, identical to the GABA
ρ1
, but has a deletion of 150 aa in the amino-terminal extracellular domain. GABA
ρ1
Δ450 mRNA injected into
Xenopus
oocytes did not produce functional GABA receptors. The second GABA
ρ1
variant (GABA
ρ1
Δ51) contains a 51-nt deletion. In
Xenopus
oocytes, GABA
ρ1
Δ51 led to the expression of GABA receptors that had the essential GABA
ρ1
characteristics of low desensitization and bicuculline resistance. Therefore, alternative splicing increases the coding potential of this gene family expressed in the human retina, but the functional diversity created by the alternative spliced forms is still not understood.
Observation of strange kinetics in protein folding
Authors: Sabelko J. , Ervin J. , Gruebele M.
Published: July 2002

Highly nonexponential folding kinetics in aqueous solution have been observed during temperature jump-induced refolding of two proteins, yeast phosphoglycerate kinase and a ubiquitin mutant. The observations are most easily interpreted in terms of downhill folding, which posits a heterogeneous ensemble of structures en route to the folded state. The data are also reconciled with exponential kinetics measured under different experimental conditions and with titration experiments indicating cooperative folding.
VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells
Authors: Cheong Kwang Ho , Zacchetti Daniele , Schneeberger Eveline E. , Simons Kai
Published: July 2002

Apical proteins are sorted and delivered from the trans-Golgi network to the plasma membrane by a mechanism involving sphingolipid–cholesterol rafts. In this paper, we report the effects of changing the levels of VIP17/MAL, a tetraspan membrane protein localized to post-Golgi transport containers and the apical cell surface in MDCK cells. Overexpression of VIP17/MAL disturbed the morphology of the MDCK cell layers by increasing apical delivery and seemingly expanding the apical cell surface domains. On the other hand, expression of antisense RNA directed against VIP17/MAL caused accumulation in the Golgi and/or impaired apical transport of different apical protein markers, i.e., influenza virus hemagglutinin, the secretory protein clusterin (gp80), the transmembrane protein gp114, and a glycosylphosphatidylinositol-anchored protein. However, antisense RNA expression did not affect the distribution of E-cadherin to the basolateral surface. Because VIP17/MAL associates with sphingolipid–cholesterol rafts, these data provide functional evidence that this protein is involved in apical transport and might be a component of the machinery clustering lipid rafts with apical cargo to form apical transport carriers.
cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain
Authors: Lund Erik G. , Guileyardo Joseph M. , Russell David W.
Published: July 2002

The turnover of cholesterol in the brain is thought to occur via conversion of excess cholesterol into 24S-hydroxycholesterol, an oxysterol that is readily secreted from the central nervous system into the plasma. To gain molecular insight into this pathway of cholesterol metabolism, we used expression cloning to isolate cDNAs that encode murine and human cholesterol 24-hydroxylases. DNA sequence analysis indicates that both proteins are localized to the endoplasmic reticulum, share 95% identity, and represent a new cytochrome P450 subfamily (CYP46). When transfected into cultured cells, the cDNAs produce an enzymatic activity that converts cholesterol into 24S-hydroxycholesterol, and to a lesser extent, 25-hydroxycholesterol. The cholesterol 24-hydroxylase gene contains 15 exons and is located on human chromosome 14q32.1. Cholesterol 24-hydroxylase is expressed predominantly in the brain as judged by RNA and protein blotting.
In situ
mRNA hybridization and immunohistochemistry localize the expression of this P450 to neurons in multiple subregions of the brain. The concentrations of 24S-hydroxycholesterol in serum are low in newborn mice, reach a peak between postnatal days 12 and 15, and thereafter decline to baseline levels. In contrast, cholesterol 24-hydroxylase protein is first detected in the brain of mice at birth and continues to accumulate with age. We conclude that the cloned cDNAs encode cholesterol 24-hydroxylases that synthesize oxysterols in neurons of the brain and that secretion of 24S-hydroxycholesterol from this tissue in the mouse is developmentally regulated.
Revised geochronology of the Casamayoran South American Land Mammal Age: Climatic and biotic implications
Authors: Kay Richard F. , Madden Richard H. , Vucetich M. Guiomar , Carlini Alfredo A. , Mazzoni Mario M. , Re Guillermo H. , Heizler Matthew , Sandeman Hamish
Published: July 2002

Isotopic age determinations (40Ar/39Ar) and associated magnetic polarity stratigraphy for Casamayoran age fauna at Gran Barranca (Chubut, Argentina) indicate that the Barrancan “subage” of the Casamayoran South American Land Mammal “Age” is late Eocene, 18 to 20 million years younger than hitherto supposed. Correlations of the radioisotopically dated magnetic polarity stratigraphy at Gran Barranca with the Cenozoic geomagnetic polarity time scale indicate that Barrancan faunal levels at the Gran Barranca date to within the magnetochronologic interval from 35.34 to 36.62 megannums (Ma) or 35.69 to 37.60 Ma. This age revision constrains the timing of an adaptive shift in mammalian herbivores toward hypsodonty. Specifically, the appearance of large numbers of hypsodont taxa in South America occurred sometime between 36 and 32 Ma (late Eocene–early Oligocene), at approximately the same time that other biotic and geologic evidence has suggested the Southern high latitudes experienced climatic cooling associated with Antarctic glaciation.
Modification of EWS/WT1 functional properties by phosphorylation
Authors: Kim Jungho , Lee Joseph M. , Branton Philip E. , Pelletier Jerry
Published: July 2002

In many human cancers, tumor-specific chromosomal rearrangements are known to create chimeric products with the ability to transform cells. The EWS/WT1 protein is such a fusion product, resulting from a t(11;22) chromosomal translocation in desmoplastic small round cell tumors, where 265 aa from the EWS amino terminus are fused to the DNA binding domain of the WT1 tumor suppressor gene. Herein, we find that EWS/WT1 is phosphorylated
in vivo
on serine and tyrosine residues and that this affects DNA binding and homodimerization. We also show that EWS/WT1 can interact with, and is a substrate for, modification on tyrosine residues by c-Abl. Tyrosine phosphorylation of EWS/WT1 by c-Abl negatively regulates its DNA binding properties. These results indicate that the biological activity of EWS/WT1 is closely linked to its phosphorylation status.
Bacterial diversity within the human subgingival crevice
Authors: Kroes Ian , Lepp Paul W. , Relman David A.
Published: July 2002

Molecular, sequence-based environmental surveys of microorganisms have revealed a large degree of previously uncharacterized diversity. However, nearly all studies of the human endogenous bacterial flora have relied on cultivation and biochemical characterization of the resident organisms. We used molecular methods to characterize the breadth of bacterial diversity within the human subgingival crevice by comparing 264 small subunit rDNA sequences from 21 clone libraries created with products amplified directly from subgingival plaque, with sequences obtained from bacteria that were cultivated from the same specimen, as well as with sequences available in public databases. The majority (52.5%) of the directly amplified 16S rRNA sequences were <99% identical to sequences within public databases. In contrast, only 21.4% of the sequences recovered from cultivated bacteria showed this degree of variability. The 16S rDNA sequences recovered by direct amplification were also more deeply divergent; 13.5% of the amplified sequences were more than 5% nonidentical to any known sequence, a level of dissimilarity that is often found between members of different genera. None of the cultivated sequences exhibited this degree of sequence dissimilarity. Finally, direct amplification of 16S rDNA yielded a more diverse view of the subgingival bacterial flora than did cultivation. Our data suggest that a significant proportion of the resident human bacterial flora remain poorly characterized, even within this well studied and familiar microbial environment.
Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1β are differentially regulated in human blood mononuclear cells and mouse spleen cells
Authors: Puren Adrian J. , Fantuzzi Giamila , Dinarello Charles A.
Published: July 2002

Interleukin (IL)-18, formerly called interferon γ (IFN-γ)-inducing factor, is biologically and structurally related to IL-1β. A comparison of gene expression, synthesis, and processing of IL-18 with that of IL-1β was made in human peripheral blood mononuclear cells (PBMCs) and in human whole blood. Similar to IL-1β, the precursor for IL-18 requires processing by caspase 1. In PBMCs, mature but not precursor IL-18 induces IFN-γ; in whole human blood stimulated with endotoxin, inhibition of caspase 1 reduces IFN-γ production by an IL-1β-independent mechanism. Unlike the precursor for IL-1β, precursor for IL-18 was expressed constitutively in PBMCs and in fresh whole blood from healthy human donors. Western blotting of endotoxin-stimulated PBMCs revealed processed IL-1β in the supernatants via an caspase 1-dependent pathway. However, in the same supernatants, only unprocessed precursor IL-18 was found. Unexpectedly, precursor IL-18 was found in freshly obtained PBMCs and constitutive IL-18 gene expression was present in whole blood of healthy donors, whereas constitutive IL-1β gene expression is absent. Similar to human PBMCs, mouse spleen cells also constitutively contained the preformed precursor for IL-18 and expressed steady-state IL-18 mRNA, but there was no IL-1β protein and no spontaneous gene expression for IL-1β in these same preparations. We conclude that although IL-18 and IL-1β are likely members of the same family, constitutive gene expression, synthesis, and processing are different for the two cytokines.
An exon that prevents transport of a mature mRNA
Authors: MacMorris Margaret A. , Zorio Diego A. R. , Blumenthal Thomas
Published: July 2002

In
Caenorhabditis elegans
, pre-mRNA for the essential splicing factor U2AF
65
sometimes is spliced to produce an RNA that includes an extra 216-bp internal exon, exon 3. Inclusion of exon 3 inserts an in-frame stop codon, yet this RNA is not subject to SMG-mediated RNA surveillance. To test whether exon 3 causes RNA to remain nuclear and thereby escape decay, we inserted it into the 3′ untranslated region of a
gfp
reporter gene. Although exon 3 did not affect accumulation or processing of the mRNA, it dramatically suppressed expression of green fluorescent protein (GFP). We showed by
in situ
hybridization that exon 3-containing
gfp
RNA is retained in the nucleus. Intriguingly, exon 3 contains 10 matches to the 8-bp 3′ splice-site consensus. We hypothesized that U2AF might recognize this octamer and thereby prevent export. This idea is supported by RNA interference experiments in which reduced levels of U2AF resulted in a small burst of
gfp
expression.
A tradeoff between immunocompetence and sexual ornamentation in domestic fowl
Authors: Verhulst S. , Dieleman S. J. , Parmentier H. K.
Published: July 2002

Females often select their mates on the basis of the size or intensity of sexual ornaments, and it is thought that such traits are reliable indicators of male quality because the costliness of these traits prevents cheating. The immunocompetence handicap hypothesis is a recently proposed mechanistic explanation of these costs and states that males carry ornaments at the expense of their resistance to disease and parasites. The tradeoff between immunocompetence and sexual ornamentation was hypothesized to arise as a consequence of the dual effect of androgens on ornamentation (+) and immune function (−). To test this hypothesis, we compared comb size between male domestic chickensGallus domesticusof lines divergently selected for antibody responses to sheep erythrocytes (three lines: selected for low response or high response and a control line). The importance of comb size in inter- and intrasexual selection is well established, and comb size is strongly dependent on testosterone level. Comb size was larger in the males of the low line than in the high line, and comb size of control males was intermediate, indicating a tradeoff between ornamentation and immunocompetence. Testosterone (T) levels varied in a similar fashion (TLow> TControl> THigh), suggesting that this hormone could mediate the tradeoff between ornamentation and immunocompetence. These results support the idea that a tradeoff with immune function may constrain the expression of secondary sexual ornaments.
An apamin-sensitive Ca
2+
-activated K
+
current in hippocampal pyramidal neurons
Authors: Stocker Martin , Krause Michael , Pedarzani Paola
Published: July 2002

In hippocampal and other cortical neurons, action potentials are followed by afterhyperpolarizations (AHPs) generated by the activation of small-conductance Ca
2+
-activated K
+
channels (SK channels). By shaping the neuronal firing pattern, these AHPs contribute to the regulation of excitability and to the encoding function of neurons. Here we report that CA1 pyramidal neurons express an AHP current that is suppressed by apamin and is involved in the control of repetitive firing. This current presents distinct kinetic and pharmacological features, and it is modulated differently than the apamin-insensitive slow AHP current. Furthermore, our
in situ
hybridizations show that the apamin-sensitive SK subunits are expressed in CA1 pyramidal neurons, providing a potential molecular correlate to the apamin-sensitive AHP current. Altogether, these results clarify the discrepancy between the reported high density of apamin-binding sites in the CA1 region and the apparent lack of an apamin-sensitive current in CA1 pyramidal neurons, and they may explain the effects of this toxin on hippocampal synaptic plasticity and learning.
Competition between a sterol biosynthetic enzyme and tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppression
Authors: Benko Ann L. , Vaduva Gabriela , Martin Nancy C. , Hopper Anita K.
Published: July 2002

The
Saccharomyces cerevisiae
Mod5 protein catalyzes isopentenylation of A to i
6
A on tRNAs in the nucleus, cytosol, and mitochondria. The substrate for Mod5p, dimethylallyl pyrophosphate, is also a substrate for Erg20p that catalyzes an essential step in sterol biosynthesis. Changing the distribution of Mod5p so that less Mod5p is present in the cytosol decreases i
6
A on cytosolic tRNAs and alters tRNA-mediated nonsense suppression. We devised a colony color/growth assay to assess tRNA-mediated nonsense suppression and used it to search for genes, which, when overexpressed, affect nonsense suppression. We identified
SAL6
,
TEF4
, and YDL219w, all of which likely affect nonsense suppression via alteration of the protein synthesis machinery. We also identified
ARC1
, whose product interacts with aminoacyl synthetases. Interestingly, we identified
ERG20
. Midwestern analysis showed that yeast cells overproducing Erg20p have reduced levels of i
6
A on tRNAs. Thus, Erg20p appears to affect nonsense suppression by competing with Mod5p for substrate. Identification of
ERG20
reveals that yeast have a limited pool of dimethylallyl pyrophosphate. It also demonstrates that disrupting the balance between enzymes that use dimethylallyl pyrophosphate as substrate affects translation.
IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNγ-responsive promoters in macrophages
Authors: Contursi Cristina , Wang I-Ming , Gabriele Lucia , Gadina Massimo , O'Shea John , Morse Herbert C. , Ozato Keiko
Published: July 2002

IFNγ, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNγ's functions depend on the activation of STAT1, which stimulates transcription of IFNγ-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbγ or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNγ-inducible genes in macrophages. We found that macrophages from ICSBP−/− mice were defective in inducing some IFNγ-responsive genes, even though they were capable of activating STAT1 in response to IFNγ. Accordingly, IFNγ activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP−/− macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNγ-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNγ treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNγ through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNγ's unique activities in immune cells.
Tumor necrosis factor α mediates apoptosis of brown adipocytes and defective brown adipocyte function in obesity
Authors: Nisoli Enzo , Briscini Luca , Giordano Antonio , Tonello Cristina , Wiesbrock Sarah M. , Uysal K. Teoman , Cinti Saverio , Carruba Michele O. , Hotamisligil Gökhan S.
Published: July 2002

Severe quantitative and qualitative brown adipocyte defects are common in obesity. To investigate whether aberrant expression of tumor necrosis factor α (TNF-α) in obesity is involved in functional brown fat atrophy, we have studied genetically obese (
ob
/
ob
) mice with targeted null mutations in the genes encoding the two TNF receptors. The absence of both TNF receptors or p55 receptor alone resulted in a significant reduction in brown adipocyte apoptosis and an increase in β
3
-adrenoreceptor and uncoupling protein-1 expression in obese mice. Increased numbers of multilocular functionally active brown adipocytes, and improved thermoregulation was also observed in obese animals lacking TNF-α function. These results indicate that TNF-α plays an important role in multiple aspects of brown adipose tissue biology and mediates the abnormalities that occur at this site in obesity.
A Rab11-containing rapidly recycling compartment in macrophages that promotes phagocytosis
Authors: Cox Dianne , Lee Donna J. , Dale Benjamin M. , Calafat Jero , Greenberg Steven
Published: July 2002

Macrophages are specialized cells of the immune system that exhibit a prodigious capacity for phagocytosis. The ability of macrophages to internalize a substantial proportion of their plasma membrane during phagocytosis indicates that they possess a mechanism for the rapid renewal of plasma membrane. We examined the role of endocytic membrane recycling in promoting phagocytosis. In contrast to many other cell types, macrophages lack a morphologically distinct peri-centriolar recycling compartment but instead demonstrate an extensive network of transferrin receptor-positive tubules and vesicles that participated in recycling. The rate of transferrin recycling in thioglycollate-elicited murine peritoneal macrophages (thio-macrophages) was exceedingly rapid, with exocytic rate constants that were 2- to 3-fold higher than those of most other cells. Because the GTPase Rab11 has been implicated in transferrin recycling in other cells, we determined its role in transferrin recycling and phagocytosis in macrophages. Macrophages expressing epitope-tagged Rab11 demonstrated the presence of Rab11 in several intracellular membrane compartments, including endosomes and nascent phagosomes. Expression of Rab11 25N, a GTP binding-deficient allele of Rab11, led to a decreased rate of transferrin efflux and impaired FcγR-mediated phagocytosis, where FcγR is the receptor for the Fc portion of IgG. In contrast, expression of Rab11 70L, a GTPase-deficient allele of Rab11, led to an increased rate of transferrin efflux and enhanced phagocytosis. We conclude that macrophages have adapted a rapidly mobilizable, endocytic compartment to enhance phagocytosis. Rab11 participates in the recruitment of this compartment to the macrophage cell surface.
MDM2 inhibits p300-mediated p53 acetylation and activation by forming a ternary complex with the two proteins
Authors: Kobet Eric , Zeng Xiaoya , Zhu Yong , Keller David , Lu Hua
Published: July 2002

p300 acetylates and activates the tumor suppressor p53 after DNA
damage. Here, we show that MDM2, a negative-feedback regulator of p53,
inhibited p300-mediated p53 acetylation by complexing with these two
proteins. First, we purified a p300–MDM2–p53 protein complex from
HeLa nuclear extracts, which was inactive in p53 acetylation, but
active in histone acetylation. Also, wild-type, but not N-terminally
deleted, MDM2 inhibited p53 acetylation by p300
in vitro
and
in vivo
. This inhibition was specific for p53,
because MDM2 did not affect acetylation of histones or the C terminus
of p73 by p300. Consequently, wild-type, but not the mutant, MDM2
repressed the p300-stimulated sequence-specific DNA-binding and
transcriptional activities of p53. These results demonstrate that an
additional mechanism of p53 inactivation by MDM2 is to inhibit p53
acetylation by p300.
Evolution of biological complexity
Authors: Adami Christoph , Ofria Charles , Collier Travis C.
Published: July 2002

To make a case for or against a trend in the evolution of complexity in biological evolution, complexity needs to be both rigorously defined and measurable. A recent information-theoretic (but intuitively evident) definition identifies genomic complexity with the amount of information a sequence stores about its environment. We investigate the evolution of genomic complexity in populations of digital organisms and monitor in detail the evolutionary transitions that increase complexity. We show that, because natural selection forces genomes to behave as a natural “Maxwell Demon,” within a fixed environment, genomic complexity is forced to increase.
Social Dilemmas
Authors: Dawes Robyn M. , Messick David M.
Published: July 2002

In social dilemma situations, each individual always receives a higher payoff for defecting than for cooperating, but all are better off if all cooperate than if all defect. Often, however, people in social dilemmas attend more to the group's payoffs than to their own, either automatically or to behave “appropriately.” But whereas social identity elicits cooperative behaviour in dilemmas, it is generally only for the benefit of an “in‐group.” Dilemmas between groups (requiring self‐sacrificial behaviour within) are often the most extreme. Consequently, the framing and manipulation of group identity is critical to cooperation rate as demonstrated by careful laboratory experimentation.
Effects of Social Deviance Labels on Judgements of Facial Attractiveness: A Comparison of Labelling Procedures Using Japanese Raters
Authors: Kowner Rotem
Published: July 2002

Are people labelled as social deviants also perceived as less physically attractive? Four studies conducted in Japan examined judgements of male social‐deviants' attractiveness using several labelling procedures. Study 1 determined attractiveness judgement and social distance of various social groups, and Study 2 examined the effect of three labels on attractiveness judgement of 16 portraits. In Studies 3 and 4 subjects were asked to match 8 or an unlimited number of the 16 portraits with the same 3 labels. Findings indicate that labelling a person negatively resulted in a decrease in attractiveness ratings. Overall, however, the labelling pattern was found to affect attractiveness judgement. Finally, this study provides cross‐cultural support for the stereotypical association between social deviants and low attractiveness.
City Growth as a Leap-frogging Process: An Application to the Tel-Aviv Metropolis
Authors: Benguigui Lucien , Czamanski Daniel , Marinov Maria
Published: July 2002

This paper presents an analysis of the spatial and temporal development of Tel-Aviv. The central notion in the analysis is the old concept of leap-frogging. Instead of seeing the population's evolution as a growth process from the centre of the metropolis outwards, we consider the population's development as taking place in different centres in a series of successive 'jumps'. In order to quantify the process, the growth of the centres' populations is fitted with the same mathematical function. This procedure enables us to extract the relevant parameters of each centre's growth and to make comparisons among them. We show that the population growth in the various centres is very similar. It permits the use of the scaling concept. In addition, a model of the growth of the individual centre is developed.
Globalisation, Competition and the Politics of Local Economic Development
Authors: Cox Kevin R.
Published: July 2002

The study of urban politics in the US is now dominated by issues of local economic development. These are situated analytically with respect to a more global space economy. This is the New Urban Politics. The central logic of the New Urban Politics is one of cities or communities competing for mobile capital. This competition supposedly has adverse distributional consequences for those living in cities or communities. Three areas of critical contention are focused on: the identity of the competing agents; the concept of firm competition underpinning the central idea of mobile capitals; and the distributional consequences of the competition for those capitals. Serious problems in abstraction and in the concept of capital adopted in this literature are identified.
Conceptualising and Mapping the Structure of the World System's City System
Authors: Smith David A. , Timberlake Michael
Published: July 2002

For the past 20 years, researches using the lens of world system theory (and other global political economy perspectives) have come to a better understanding of many of the anomalies in urbanisation patterns across more and less developed countries that had befuddled researchers whose assumptions left out global sources of social change. Recently this line of research has moved beyond regarding cities as mere objects of global forces, also theorising about their importance as lynchpins in the spatial organisation of the world economy. In this paper we review some of the scholarship that emphasises large cities' roles as important modes of production, consumption, exchange and control at the global level; we develop the argument that systematic tinkages—economic, cultural, political or social-retational—among global cities are likely to reveal the spatial organisation of the world-system; we review our position that formal network analysis provides a most promising methodological framework for analysing and mapping global intercity linkages; and we present a map of the current world city system based on our network analysis of recent air travel among many of the world's great cities. We point out that our analysis is very preliminary and provides only a rough chart of the world city system at one point in time, and the data requirements for more detailed world city system maps for several periods of time are imposing, to say the least. Nevertheless, such a project holds the promise of revealing much about the spatial structure of our world system, how it has changed; how it is likely to change in the future; and how cities' populations are affected by these changes. Completing the project will probably require collaboration among researchers in different countries.