bluuebunny/crossref_metadata_2025 huggingface dataset:
A new prostaglandin E receptor mediates calcium influx and acrosome reaction in human spermatozoa
Authors: Schaefer Michael , Hofmann Thomas , Schultz Günter , Gudermann Thomas
Published: July 2002

Zona pellucida protein 3, a protein of the egg’s extracellular matrix, and progesterone secreted by granulosa cells surrounding the oocyte are regarded as physiological stimuli of sperm acrosome reaction. Signal transduction steps initiated by both stimuli result in influx of Ca
2+
from the extracellular space. Herein, we propose a role for prostaglandin (PG) E as a physiological inducer of Ca
2+
influx and acrosome reaction in human spermatozoa. PGE
1
specifically binds to human sperm membranes (
K
d
= 20.4 nM;
B
max
= 88 fmol/mg protein) and induces a pertussis toxin-insensitive, transient increase in intracellular Ca
2+
concentrations, which can be blocked by μM concentrations of La
3+
, Gd
3+
, and Zn
2+
. The kinetic profile was similar to that observed after progesterone challenge. Sequential application of both agonists did not lead to cross-desensitization. E prostaglandins were found to be the only prostanoids with agonistic properties (EC
50
values for PGE
1
and PGE
2
: <10 nM and 300 nM, respectively). Pharmacological characteristics were not compatible with those of cloned prostanoid receptors indicating the expression of a distinct membrane receptor. Activation of the sperm E prostanoid receptor stimulates incorporation of [α-
32
P]GTP azidoanilide into immunoprecipitated Gα
q/11
subunits. Thus, in human sperm, PG induces Ca
2+
influx and acrosome reaction via a G
q/11
-coupled E prostanoid receptor. The block of PGE
1
-induced Ca
2+
transients and acrosome reaction by physiological Zn
2+
concentrations highlights a role of Zn
2+
as an endogenous Ca
2+
channel blocker present in seminal plasma protecting sperm from premature PGE
1
-evoked increases in intracellular Ca
2+
concentrations.
Crystal structure of the catalytic domain of human tumor necrosis factor-α-converting enzyme
Authors: Maskos Klaus , Fernandez-Catalan Carlos , Huber Robert , Bourenkov Gleb P. , Bartunik Hans , Ellestad George A. , Reddy Pranitha , Wolfson Martin F. , Rauch Charles T. , Castner Beverly J. , Davis Raymond , Clarke Howard R. G. , Petersen Melissa , Fitzner Jeffrey N. , Cerretti Douglas Pat , March Carl J. , Paxton Raymond J. , Black Roy A. , Bode Wolfram
Published: July 2002

Tumor necrosis factor-α (TNFα) is a cytokine that induces protective inflammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNFα is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNFα-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 Å crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin/ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNFα cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNFα cone. The active-site cleft of TACE shares properties with the matrix metalloproteinases but exhibits unique features such as a deep S3′ pocket merging with the S1′ specificity pocket below the surface. The structure thus opens a different approach toward the design of specific synthetic TACE inhibitors, which could act as effective therapeutic agents
in vivo
to modulate TNFα-induced pathophysiological effects, and might also help to control related shedding processes.
Telomere loss in cells treated with cisplatin
Authors: Ishibashi Toru , Lippard Stephen J.
Published: July 2002

Telomeres play an important role in the immortalization of proliferating cells. The long tandem repeats of 5′-TTAGGG-3′ sequences in human telomeres are potential targets for the anticancer drug cisplatin, which forms mainly intrastrand d(GpG) and d(ApG) cross-links on DNA. The present study reveals that telomeres in cisplatin-treated HeLa cells are markedly shortened and degraded. A dose that killed 61% of the cells but allowed one round of cell division resulted in shortened telomeres before the induction of apoptosis. Higher doses of cisplatin halted cell cycle progression during the first S phase and triggered apoptosis followed by degradation of telomere repeats. A model in which both cell division with incomplete replication and induction of apoptosis by cisplatin could occur was devised to explain the drug-induced telomere loss.
Bcl-X
L
interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation
Authors: Hu Yuanming , Benedict Mary A. , Wu Dayang , Inohara Naohiro , Núñez Gabriel
Published: July 2002

Recent studies indicate that
Caenorhabditis elegans
CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9
in vivo
. Bcl-X
L
, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-X
L
was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-X
L
inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-X
L
purified from
Escherichia coli
or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-X
L
failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-X
L
regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-X
L
associates with caspase-9 and Apaf-1, and show that Bcl-X
L
inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.
Extent of genomic rearrangement after genome duplication in yeast
Authors: Seoighe Cathal , Wolfe Kenneth H.
Published: July 2002

Whole-genome duplication approximately 108years ago was proposed as an explanation for the many duplicated chromosomal regions inSaccharomyces cerevisiae. Here we have used computer simulations and analytic methods to estimate some parameters describing the evolution of the yeast genome after this duplication event. Computer simulation of a model in which 8% of the original genes were retained in duplicate after genome duplication, and 70–100 reciprocal translocations occurred between chromosomes, produced arrangements of duplicated chromosomal regions very similar to the map of real duplications in yeast. An analytical method produced an independent estimate of 84 map disruptions. These results imply that many smaller duplicated chromosomal regions exist in the yeast genome in addition to the 55 originally reported. We also examined the possibility of determining the original order of chromosomal blocks in the ancestral unduplicated genome, but this cannot be done without information from one or more additional species. If the genome sequence of one other species (such asKluyveromyces lactis) were known it should be possible to identify 150–200 paired regions covering the whole yeast genome and to reconstruct approximately two-thirds of the original order of blocks of genes in yeast. Rates of interchromosome translocation in yeast and mammals appear similar despite their very different rates of homologous recombination per kilobase.
An arrayable flow-through microcentrifuge for high-throughput instrumentation
Authors: Marziali Andre , Willis Thomas D. , Davis Ronald W.
Published: July 2002

A compact, flow-through centrifugation system has been developed specifically for high-throughput centrifugation of large numbers of samples. The instrument is based on multiple high-speed rotors that also serve as sample holders. The small size of the rotors allows them to be arrayed in a standard 96-well microtiter plate spacing, making this device ideal for highly parallel automated instrumentation. Though initially designed for cell separation in DNA sequencing protocols, the flow-through centrifuge can be used to replace conventional centrifugation in most processes involving small samples. Techniques for recovery of both the supernatant and the pellet have been developed, as well as techniques for sample mixing, and cleaning of the reusable rotors. This paper discusses the design and performance of the flow-through centrifuge applied to cell separation and resuspension and to DNA purification and concentration.
Solutions of a Lagrangian system on 𝕋
2
Authors: Rabinowitz Paul H.
Published: July 2002

A Lagrangian system on 𝕋
2
that has been studied earlier under a geometrical condition and found to possess a pair of solutions,

H
±

, homoclinic to periodic solutions,

v
±

, of a given homotopy type, is considered further. It is shown with the aid of

H
±

and variational arguments that, in fact, there is a much richer structure of homoclinics and heteroclinics to

v
±

. Indeed, the system admits chaotic solutions.
Separation of C/EBPα-mediated proliferation arrest and differentiation pathways
Authors: Müller Christine , Alunni-Fabbroni Marianna , Kowenz-Leutz Elisabeth , Mo Xianming , Tommasino Massimo , Leutz Achim
Published: July 2002

Cell proliferation and terminal differentiation are mutually exclusive in most cell lineages. The b-zip transcription factor CCAAT/enhancer-binding protein α (C/EBPα) induces proliferation arrest and differentiation in many cell types, suggesting that both activities are linked. Here we show that C/EBPα-mediated proliferation arrest and differentiation pathways can be separated by the E7 oncoprotein of the “high-risk” human papilloma virus 16. The E7 oncoprotein overrides C/EBPα-mediated cell cycle withdrawal without compromising the transactivation activity of C/EBPα or its ability to participate in differentiation. Uncoupling of both pathways depends on the casein kinase II site of the oncoprotein but not on its ability to neutralize pocket proteins or the cyclin-dependent kinase inhibitor protein p21. Our results suggest a bifurcation of C/EBPα-mediated proliferation arrest and differentiation pathways.
Distinct neurochemical features of acute and persistent pain
Authors: Basbaum Allan I.
Published: July 2002

To address the neurochemistry of the mechanisms that underlie the
development of acute and persistent pain, our laboratory has been
studying mice with deletions of gene products that have been implicated
in nociceptive processing. We have recently raised mice with a deletion
of the preprotachykinin-A gene, which encodes the peptides substance P
(SP) and neurokinin A (NKA). These studies have identified a specific
behavioral phenotype in which the animals do not detect a window of
“pain” intensities; this window cuts across thermal, mechanical,
and chemical modalities. The lowered thermal and mechanical withdrawal
thresholds that are produced by tissue or nerve injury, however, were
still present in the mutant mice. Thus, the behavioral manifestations
of threshold changes in nociceptive processing in the setting of injury
do not appear to require SP or NKA. To identify relevant neurochemical
factors downstream of the primary afferent, we are also studying the
dorsal horn second messenger systems that underlie the development of
tissue and nerve injury-induced persistent pain states. We have
recently implicated the γ isoform of protein kinase C (PKCγ) in the
development of nerve injury-induced neuropathic pain. Acute pain
processing, by contrast, is intact in the PKCγ-null mice. Taken
together, these studies emphasize that there is a distinct
neurochemistry of acute and persistent pain. Persistent pain should be
considered a disease state of the nervous system, not merely a
prolonged acute pain symptom of some other disease conditions.
DIO-1is a gene involved in onset of apoptosisin vitro,whose misexpression disrupts limb development
Authors: García-Domingo David , Leonardo Esther , Grandien Alf , Martínez Pedro , Albar Juan Pablo , Izpisúa-Belmonte Juan Carlos , Martínez-A Carlos
Published: July 2002

TheDIO-1(death inducer-obliterator-1) gene, identified by differential display PCR in pre-B WOL-1 cells undergoing apoptosis, encodes a putative transcription factor whose protein has two Zn finger motifs, nuclear localization signals, and transcriptional activation domains, expressed in the limb interdigitating webs during development. When overexpressed,DIO-1translocates to the nucleus and activates apoptosisin vitro. Nuclear translocation as well as induction of apoptosis are lost after deletion of the nuclear localization sequences.DIO-1apoptotic induction is prevented by caspase inhibitors and Bcl-2 overexpression. Thein vivorole ofDIO-1was studied by misexpressingDIO-1during chicken limb development. The most frequently observed phenotype was an arrest in limb outgrowth, an effect that correlates with the inhibition of mesodermal and ectodermal genes involved in this process. Our data demonstrate the ability ofDIO-1to trigger apoptotic processesin vitroand suggest a role for this gene in cell death during development.
Dichotomy between naïve and memory CD4+T cell responses to Fas engagement
Authors: Desbarats J. , Wade T. , Wade W. F. , Newell M. K.
Published: July 2002

Engagement of Fas (APO-1, CD95), a member of the tumor necrosis factor receptor superfamily, can induce apoptotic cell death. However, Fas engagement also can costimulate lymphocyte proliferation. The physiologic regulation of these two outcomes is poorly understood. Here, we have used two systems, the firstin vitroand the secondin vivo, to demonstrate that naïve and memory CD4+T cells display dichotomous responses to Fas ligation. Naïve CD4+T cells (CD44lo, CD45RB+, CD62L+) die as a consequence of Fas ligation in the presence of anti-CD3 antibody, whereas memory T cells (CD44hi, CD45RB−, CD62L−), freshly isolated from the same starting population and subjected to the same stimulation conditions, are costimulated to proliferate by Fas ligation.In vitro, we demonstrate that CD28-mediated signals or T helper 1 and T helper 2 differentiation cytokines alter the response of naïve T cells, but not of memory T cells, to Fas ligation.In vivoexperiments in hen egg lysozyme (HEL) T cell receptor transgenic mice show that CD4+T cells from HEL-naïve mice are killed by Fas ligation, but CD4+T cells from long-term HEL-exposed mice are costimulated by Fas ligation. Thus, the physiological outcome of Fas ligation in CD4+T cells is determined primarily by the antigenic history of the T cell.
Cell cycle regulation of Dfp1, an activator of the Hsk1 protein kinase
Authors: Brown Grant W. , Kelly Thomas J.
Published: July 2002

In fission yeast, the Hsk1 protein kinase is essential for the initiation of DNA replication. We have shown previously that Hsk1 forms a heterodimeric complex with the regulatory subunit, Dfp1. In this report we describe the further characterization of Dfp1. Reconstitution experiments with purified proteins indicate that Dfp1 is necessary and sufficient to activate Hsk1 phosphorylation of exogenous substrates, such as the
Schizosaccharomyces pombe
minichromosome maintenance protein Cdc19. The
dfp1
+
gene is essential for viability of
S. pombe
, and depletion of the Dfp1 protein significantly delays the onset of S phase. Dfp1 is a phosphoprotein
in vivo
and becomes hyperphosphorylated when cells are blocked in S phase by treatment with the DNA synthesis inhibitor hydroxyurea. Hyperphosphorylation in S phase depends on the checkpoint kinase Cds1. The abundance of Dfp1 varies during progression through the cell cycle. The protein is absent when cells are arrested in G
1
phase. When cells are released into the cell cycle, Dfp1 appears suddenly at the G
1
/S transition, coincident with the initiation of DNA replication. The absence of Dfp1 before S phase is due largely, but not exclusively, to posttranscriptional regulation. We propose that cell cycle-regulated activation of Dfp1 expression at the G
1
/S transition results in activation of the Hsk1 protein kinase, which, in turn, leads to the initiation of DNA replication.
A systematic study of low-resolution recognition in protein–protein complexes
Authors: Vakser Ilya A. , Matar Omar G. , Lam Chan F.
Published: July 2002

A comprehensive nonredundant database of 475 cocrystallized protein–protein complexes was used to study low-resolution recognition, which was reported in earlier docking experiments with a small number of proteins. The docking program
gramm
was used to delete the atom-size structural details and systematically dock the resulting molecular images. The results reveal the existence of the low-resolution recognition in 52% of all complexes in the database and in 76% of the 113 complexes with an interface area >4,000 Å
2
. Limitations of the docking and analysis tools used in this study suggest that the actual number of complexes with the low-resolution recognition is higher. However, the results already prove the existence of the low-resolution recognition on a broad scale.
Long-term regulated expression of growth hormone in mice after intramuscular gene transfer
Authors: Rivera Victor M. , Ye Xuehai , Courage Nancy L. , Sachar Jeff , Cerasoli Frank , Wilson James M. , Gilman Michael
Published: July 2002

Effective delivery of secreted proteins by gene therapy will require a vector that directs stable delivery of a transgene and a regulatory system that permits pharmacologic control over the level and kinetics of therapeutic protein expression. We previously described a regulatory system that enables transcription of a target gene to be controlled by rapamycin, an orally bioavailable drug. Here we demonstrate
in vivo
regulation of gene expression after intramuscular injection of two separate adenovirus or adeno-associated virus (AAV) vectors, one encoding an inducible human growth hormone (hGH) target gene, and the other a bipartite rapamycin-regulated transcription factor. Upon delivery of either vector system into immunodeficient mice, basal plasma hGH expression was undetectable and was induced to high levels after administration of rapamycin. The precise level and duration of hGH expression could be controlled by the rapamycin dosing regimen. Equivalent profiles of induction were observed after repeated administration of single doses of rapamycin over many months. AAV conferred stable expression of regulated hGH in both immunocompetent and immunodeficient mice, whereas adenovirus-directed hGH expression quickly extinguished in immunocompetent animals. These studies demonstrate that the rapamycin-based regulatory system, delivered intramuscularly by AAV, fulfills many of the conditions necessary for the safe and effective delivery of therapeutic proteins by gene therapy.
General covariance and harmonic maps
Authors: Sternberg Shlomo
Published: July 2002

The principle of general covariance is used to derive the conditions for a map to be harmonic. Modification to the harmonic map equations due to the presence of a Yang–Mills field is described.
Genome signature comparisons among prokaryote, plasmid, and mitochondrial DNA
Authors: Campbell Allan , Mrázek Jan , Karlin Samuel
Published: July 2002

Our basic observation is that each genome has a characteristic “signature” defined as the ratios between the observed dinucleotide frequencies and the frequencies expected if neighbors were chosen at random (dinucleotide relative abundances). The remarkable fact is that the signature is relatively constant throughout the genome; i.e., the patterns and levels of dinucleotide relative abundances of every 50-kb segment of the genome are about the same. Comparison of the signatures of different genomes provides a measure of similarity which has the advantage that it looks at all the DNA of an organism and does not depend on the ability to align homologous sequences of specific genes. Genome signature comparisons show that plasmids, both specialized and broad-range, and their hosts have substantially compatible (similar) genome signatures. Mammalian mitochondrial (Mt) genomes are very similar, and animal and fungal Mt are generally moderately similar, but they diverge significantly from plant and protist Mt sets. Moreover, Mt genome signature differences between species parallel the corresponding nuclear genome signature differences, despite large differences between Mt and host nuclear signatures. In signature terms, we find that the archaea are not a coherent clade. For example,
Sulfolobus
and
Halobacterium
are extremely divergent. There is no consistent pattern of signature differences among thermophiles. More generally, grouping prokaryotes by environmental criteria (e.g., habitat propensities, osmolarity tolerance, chemical conditions) reveals no correlations in genome signature.
A high-density physical map of
Sinorhizobium meliloti
1021 chromosome derived from bacterial artificial chromosome library
Authors: Capela Delphine , Barloy-Hubler Frédérique , Gatius Marie-Thérèse , Gouzy Jérôme , Galibert Francis
Published: July 2002

As part of the European
Sinorhizobium meliloti
(strain 1021) chromosome sequencing project, four genomic bacterial artificial chromosome (BAC) libraries have been constructed, one of which was mainly used for chromosome mapping. This library consists of 1,824 clones with an average insert size of 80 kilobases and represents approximately 20-fold total genome coverage [6.8 megabases (Mbs)]. PCR screening of 384 BAC clones with 447 chromosomal markers (PCR primer pairs), consisting of 73 markers representing 118 genes (40 individual genes and 78 genes clustered in 23 operons), two markers from the
rrn
operon (three loci), four markers from insertion sequences (≈16 loci) and 368 sequence-tagged sites allowed the identification of 252 chromosomal BAC clones and the construction of a high-density physical map of the whole 3.7-Mb chromosome of
S. meliloti
. An average of 5.5 overlapping and colinear BAC clones per marker, correlated with a low rate of deleted or rearranged clones (0.8%) indicate a solid BAC contigation and a correct mapping. Systematic
blastx
analysis of sequence-tagged site marker sequences allowed prediction of a biological function for a number of putative ORFs. Results are available at
http://www-recomgen.univ-rennes1.fr/meliloti
. This map, whose resolution averages one marker every 9 kilobases, should provide a valuable tool for further sequencing, functional analysis, and positional cloning.
Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in
Streptomyces
avermitilis
Authors: Ikeda Haruo , Nonomiya Tomoko , Usami Masayo , Ohta Toshio , Ōmura Satoshi
Published: July 2002

Analysis of the gene cluster from
Streptomyces avermitilis
that governs the biosynthesis of the polyketide anthelmintic avermectin revealed that it contains four large ORFs encoding giant multifunctional polypeptides of the avermectin polyketide synthase (AVES 1, AVES 2, AVES 3, and AVES 4). These clustered polyketide synthase genes responsible for avermectin biosynthesis together encode 12 homologous sets of enzyme activities (modules), each catalyzing a specific round of polyketide chain elongation. The clustered genes encoding polyketide synthase are organized as two sets of six modular repeats,
aveA1-aveA2
and
aveA3-aveA4
, which are convergently transcribed. The total of 55 constituent active sites makes this the most complex multifunctional enzyme system identified to date. The sequenced DNA region contains 14 additional ORFs, some of which encode polypeptides governing other key steps in avermectin biosynthesis. Between the two sets of polyketide synthase genes lie two genes involved in postpolyketide modification, one of which encodes cynthochrome P450 hydroxylase that probably catalyzes furan ring formation at C6 to C8a. Immediately right of the large polyketide synthase genes is a set of genes involved in oleandrose biosynthesis and its transglycosylation to polyketide-derived aglycons. This cluster includes nine genes, but one is not functional in the biosynthesis of avermectin. On the left side of polyketide synthase genes, two ORFs encoding methyltransferase and nonpolyketide synthase ketoreductase involved in postpolyketide modification are located to the left of the polyketide synthase genes, and an adjacent gene encodes a regulatory function that may be involved in activation of the transcription of avermectin biosynthetic genes.
Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina
Authors: Bennett Jean , Maguire Albert M. , Cideciyan Artur V. , Schnell Michael , Glover Ernest , Anand Vibha , Aleman Tomas S. , Chirmule Narendra , Gupta Abha R. , Huang Yijun , Gao Guang-Ping , Nyberg William C. , Tazelaar John , Hughes Joseph , Wilson James M. , Jacobson Samuel G.
Published: July 2002

Recombinant adeno-associated virus (rAAV) is a promising vector for therapy of retinal degenerative diseases. We evaluated the efficiency, cellular specificity, and safety of retinal cell transduction in nonhuman primates after subretinal delivery of an rAAV carrying a cDNA encoding green fluorescent protein (EGFP), rAAV.CMV.
EGFP
. The treatment results in efficient and stable
EGFP
expression lasting >1 year. Transgene expression in the neural retina is limited exclusively to rod photoreceptors. There is neither electroretinographic nor histologic evidence of photoreceptor toxicity. Despite significant serum antibody responses to the vector, subretinal readministration results in additional transduction events. The findings further characterize the retinal cell tropism of rAAV. They also support the development of studies aimed ultimately at treating inherited retinal degeneration by using rAAV-mediated gene therapy.
Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites
Authors: Bassi Roberto , Croce Roberta , Cugini Daniela , Sandonà Dorianna
Published: July 2002

The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues. Overexpression of the apoproteins in bacteria was followed by reconstitutionin vitrowith purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites. Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a–Chl-b specificity. Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins.
Geologic constraints on clandestine nuclear testing in South Asia
Authors: Davis Dan M. , Sykes Lynn R.
Published: July 2002

Cavity decoupling in salt is the most plausible means by which a nation could conduct clandestine testing of militarily significant nuclear weapons. The conditions under which solution-mined salt can be used for this purpose are quite restrictive. The salt must be thick and reasonably pure. Containment of explosions sets a shallow limit on depth, and cavity stability sets a deep limit. These constraints are met in considerably <1% of the total land area of India and Pakistan. Most of that area is too dry for cavity construction by solution mining; disposal of brine in rivers can be detected easily. Salt domes, the most favorable structures for constructing large cavities, are not present in India and Pakistan. Confidence that they are adhering to the Comprehensive Test Ban Treaty (CTBT) is enhanced by their geological conditions, which are quite favorable to verification, not evasion. Thus, their participation in the CTBT is constrained overwhelmingly by political, not scientific, issues. Confidence in the verification of the CTBT could be enhanced if India and Pakistan permitted stations of the various monitoring technologies that are now widely deployed elsewhere to be operated on their territories.
Activation changes the spectrum but not the diversity of genes expressed by T cells
Authors: Teague T. Kent , Hildeman David , Kedl Ross M. , Mitchell Tom , Rees William , Schaefer Brian C. , Bender Jeremy , Kappler John , Marrack Philippa
Published: July 2002

During activation T cells are thought to change their patterns of gene expression dramatically. To find out whether this is true for T cells activated in animals, the patterns of genes expressed in resting T cells and T cells 8 and 48 hr after activation were examined by using Affymetrix gene arrays. Gene arrays gave accurate comparisons of gene expression in the different cell types because the expression of genes known to vary during activation changed as expected. Of the approximately 6,300 genes assessed by the arrays, about one-third were expressed to appreciable extents in any of the T cells tested. Thus, resting T cells express a surprisingly large diversity of genes. The patterns of gene expression changed considerably within 8 hr of T cell activation but returned to a disposition more like that of resting T cells within 48 hr of exposure to antigen. Not unexpectedly, the activated T cells expressed genes associated with cell division at higher levels than resting T cells. The resting T cells expressed a number of cytokine receptor genes and some genes thought to suppress cell division, suggesting that the state of resting T cells is not a passive failure to respond to extant external stimuli.
The structural basis for the oriented assembly of a TBP/TFB/promoter complex
Authors: Littlefield Otis , Korkhin Yakov , Sigler Paul B.
Published: July 2002

Recently the definition of the metazoan RNA polymerase II and
archaeal core promoters has been expanded to include a region
immediately upstream of the TATA box called the B recognition element
(BRE), so named because eukaryal transcription factor TFIIB and its
archaeal orthologue TFB interact with the element in a
sequence-specific manner. Here we present the 2.4-Å crystal structure
of archaeal TBP and the C-terminal core of TFB (TFB
c
) in a
complex with an extended TATA-box-containing promoter that provides a
detailed picture of the stereospecific interactions between the BRE and
a helix–turn–helix motif in the C-terminal cyclin repeat of
TFB
c
. This interaction is important in determining the
level of basal transcription and explicitly defines the direction of
transcription.
Reduced facilitation and vesicular uptake in crustacean and mammalian neuromuscular junction by T-588, a neuroprotective compound
Authors: Hirata Kazunari , Nakagawa Masaya , Urbano Francisco J. , Rosato-Siri Marcelo D. , Moreira Jorge E. , Uchitel Osvaldo D. , Sugimori Mutsuyuki , Llinás Rodolfo
Published: July 2002

Bath application of compound T-588, a neuroprotective agent, reduced paired-pulse and repetitive-pulse facilitation at mammalian and crustacean neuromuscular junctions. In addition, it reduced voltage-gated sodium and potassium currents in a use-dependent fashion, but had only a small effect on the presynaptic Ca
2+
conductance. By contrast, it blocked FM 1–43 vesicular uptake but not its release, in both species. Postsynaptically, T-588 reduced acetylcholine currents at the mammalian junction in a voltage-independent manner, but had no effect on the crayfish glutamate junction. All of these effects were rapidly reversible and were observed at concentrations close to the compound’s acute protective level. We propose that this set of mechanisms, which reduces high-frequency synaptic transmission, is an important contributory factor in the neuroprotective action of T-588.
Extracellular antigen processing and presentation by immature dendritic cells
Authors: Santambrogio Laura , Sato Aaron K. , Carven Gregory J. , Belyanskaya Svetlana L. , Strominger Jack L. , Stern Lawrence J.
Published: July 2002

In antigen presentation to CD4+T cells, proteins are degraded to peptide fragments and loaded onto class II MHC molecules in a process involving the peptide exchange factors H-2M (murine) or HLA-DM (human). In many antigen-presenting cells these processes occur in intracellular endosomal compartments, where peptides are generated and loaded onto class II MHC proteins for subsequent transport to the surface and presentation to T cells. Here, we provide evidence for an additional antigen-processing pathway in immature dendritic cells (DC). Immature DC express at the cell surface empty or peptide-receptive class II MHC molecules, as well as H-2M or HLA-DM. Secreted DC proteases act extracellularly to process intact proteins into antigenic peptides. Peptides produced by such activity are efficiently loaded onto cell surface class II MHC molecules. Together these elements comprise an unusual extracellular presentation pathway in which antigen processing and peptide loading can occur entirely outside of the cell.
Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic
Escherichia
coli
Authors: Sperandio Vanessa , Mellies Jay L. , Nguyen William , Shin Sooan , Kaper James B.
Published: July 2002

Enterohemorrhagic
Escherichia coli
O157:H7 and enteropathogenic
E. coli
cause a characteristic histopathology in intestinal cells known as attaching and effacing. The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Using
lacZ
reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic
E. coli
and enteropathogenic
E. coli
. The
luxS
gene recently shown to be responsible for production of autoinducer in the
Vibrio harveyi
and
E. coli
quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the
luxS
gene. Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms. These results suggest that intestinal colonization by
E. coli
O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic
E. coli
of the normal intestinal flora.
Association of HFE protein with transferrin receptor in crypt enterocytes of human duodenum
Authors: Waheed Abdul , Parkkila Seppo , Saarnio Juha , Fleming Robert E. , Zhou Xiao Yan , Tomatsu Shunji , Britton Robert S. , Bacon Bruce R. , Sly William S.
Published: July 2002

In hereditary hemochromatosis (HH), intestinal absorption of
dietary iron is increased, leading to excessive iron accumulation in
tissues and resultant organ damage. The HFE protein, which is defective
in HH, normally is expressed in crypt enterocytes of the duodenum where
it has a unique, predominantly intracellular localization. In placenta,
the HFE protein colocalizes with and forms a stable association with
the transferrin receptor (TfR), providing a link between the HFE
protein and iron transport. In the present study, we examined the
relationship of the HFE protein to the TfR in enterocytes of the human
duodenum and measured the uptake of transferrin-bound iron and ionic
iron by isolated crypt and villus enterocytes. Immunocytochemistry
showed that the HFE protein and TfR both are expressed in the crypt
enterocytes. Western blots showed that, as was the case in human
placenta, the HFE protein in crypt enterocytes is physically associated
with the TfR and with β
2
-microglobulin. The crypt cell
fraction exhibited dramatically higher transferrin-bound iron uptake
than villus cells. On the other hand, the villus cells showed 2–3
times higher uptake of ionic iron than crypt cells. We propose that the
HFE protein modulates the uptake of transferrin-bound iron from plasma
by crypt enterocytes and participates in the mechanism by which the
crypt enterocytes sense the level of body iron stores. Impairment of
this function caused by
HFE
gene mutations in HH could
provide a paradoxical signal in crypt enterocytes that programs the
differentiating enterocytes to absorb more dietary iron when they
mature into villus enterocytes.
Molecular studies suggest that cartilaginous fishes have a terminal position in the piscine tree
Authors: Rasmussen Ann-Sofie , Arnason Ulfur
Published: July 2002

The Chondrichthyes (cartilaginous fishes) are commonly accepted as being sister group to the other extant Gnathostomata (jawed vertebrates). To clarify gnathostome relationships and to aid in resolving and dating the major piscine divergences, we have sequenced the complete mtDNA of the starry skate and have included it in phylogenetic analysis along with three squalomorph chondrichthyans—the common dogfish, the spiny dogfish, and the star spotted dogfish—and a number of bony fishes and amniotes. The direction of evolution within the gnathostome tree was established by rooting it with the most closely related non-gnathostome outgroup, the sea lamprey, as well as with some more distantly related taxa. The analyses placed the chondrichthyans in a terminal position in the piscine tree. These findings, which also suggest that the origin of the amniote lineage is older than the age of the oldest extant bony fishes (the lungfishes), challenge the evolutionary direction of several morphological characters that have been used in reconstructing gnathostome relationships. Applying as a calibration point the age of the oldest lungfish fossils, 400 million years, the molecular estimate placed the squalomorph/batomorph divergence at ≈190 million years before present. This dating is consistent with the occurrence of the earliest batomorph (skates and rays) fossils in the paleontological record. The split between gnathostome fishes and the amniote lineage was dated at ≈420 million years before present.
Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells
Authors: Efstathiou J. A. , Liu D. , Wheeler J. M. D. , Kim H. C. , Beck N. E. , Ilyas M. , Karayiannakis A. J. , Mortensen N. J. McC. , Kmiot W. , Playford R. J. , Pignatelli M. , Bodmer W. F.
Published: July 2002

Epithelial (E)-cadherin and its associated cytoplasmic proteins (α-, β-, and γ-catenins) are important mediators of epithelial cell–cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (
CDH1
) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin–catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.
Induction of autoantibodies to mouse CCR5 with recombinant papillomavirus particles
Authors: Chackerian Bryce , Lowy Douglas R. , Schiller John T.
Published: July 2002

The vertebrate immune system has evolved to respond vigorously to microbial infection but to ignore self-antigens. Evidence has emerged that B cell responses to viruses are initiated by immune recognition of ordered arrays of antigen on the viral surface. To test whether autoantibodies against a self-antigen can be induced by placing it in a context that mimics the ordered surface of a viral particle, a peptide representing an extracellular loop of the mouse chemokine receptor CCR5 was incorporated into an immunodominant site of the bovine papillomavirus virus L1 coat protein, which self-assembles into virus-like particles. Mice inoculated with chimeric L1-CCR5 particles generated autoantibodies that bound to native mouse CCR5, inhibited binding of its ligand RANTES, and blocked HIV-1 infection of an indicator cell line expressing a human-mouse CCR5 chimera. These results suggest a general method for inducing autoantibodies against self-antigens, with diverse potential basic research and clinical applications.
Modulation of the baboon (
Papio anubis
) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity
Authors: Fazleabas Asgerally T. , Donnelly Kathleen M. , Srinivasan Sudha , Fortman Jeffrey D. , Miller Josephine B.
Published: July 2002

This study was undertaken to determine the modulation of uterine function by chorionic gonadotrophin (CG) in a nonhuman primate. Infusion of recombinant human CG (hCG) between days 6 and 10 post ovulation initiated the endoreplication of the uterine surface epithelium to form distinct epithelial plaques. These plaque cells stained intensely for cytokeratin and the proliferating cell nuclear antigen. The stromal fibroblasts below the epithelial plaques stained positively for α-smooth muscle actin (αSMA). Expression of αSMA is associated with the initiation of decidualization in the baboon endometrium. Synthesis of the glandular secretory protein glycodelin, as assessed by Western blot analysis, was markedly up-regulated by hCG, and this increase was confirmed by immunocytochemistry, Northern blot analysis, and reverse transcriptase-PCR. To determine whether hCG directly modulated these uterine responses, we treated ovariectomized baboons sequentially with estradiol and progesterone to mimic the hormonal profile of the normal menstrual cycle. Infusion of hCG into the oviduct of steroid-hormone-treated ovariectomized baboons induced the expression of αSMA in the stromal cells and glycodelin in the glandular epithelium. The epithelial plaque reaction, however, was not readily evident. These studies demonstrate a physiological effect of CG on the uterine endometrium
in vivo
and suggest that the primate blastocyst signal, like the blastocyst signals of other species, modulates the uterine environment prior to implantation.
Characterization of Sam68-like mammalian proteins SLM-1 and SLM-2: SLM-1 is a Src substrate during mitosis
Authors: Di Fruscio Marco , Chen Taiping , Richard Stéphane
Published: July 2002

Sam68, the 68-kDa
S
rc substrate
a
ssociated during
m
itosis, is an RNA-binding protein with signaling properties that contains a GSG (
G
RP33,
S
am68,
G
LD-1) domain. Here we report the cloning of two
S
am68-
l
ike-
m
ammalian proteins, SLM-1 and SLM-2. These proteins have an ≈70% sequence identity with Sam68 in their GSG domain. SLM-1 and SLM-2 have the characteristic Sam68 SH2 and SH3 domain binding sites. SLM-1 is an RNA-binding protein that is tyrosine phosphorylated by Src during mitosis. SLM-1 bound the SH2 and SH3 domains of p59
fyn
, Grb-2, phospholipase Cγ-1 (PLCγ-1), and/or p120
rasGAP
, suggesting it may function as a multifunctional adapter protein for Src during mitosis. SLM-2 is an RNA-binding protein that is not tyrosine phosphorylated by Src or p59
fyn
. Moreover, SLM-2 did not associate with the SH3 domains of p59
fyn
, Grb-2, PLCγ-1, or p120
rasGAP
, suggesting that SLM-2 may not function as an adapter protein for these proteins. The identification of SLM-1 and SLM-2 demonstrates the presence of a Sam68/SLM family whose members have the potential to link signaling pathways with RNA metabolism.
Genomic evolution during a 10,000-generation experiment with bacteria
Authors: Papadopoulos Dimitri , Schneider Dominique , Meier-Eiss Jessica , Arber Werner , Lenski Richard E. , Blot Michel
Published: July 2002

Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of
Escherichia coli
evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.