bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics of the interaction between a fibronectin molecule and a living bacterium under mechanical force
Authors: Bustanji Yasser , Arciola Carla Renata , Conti Matteo , Mandello Enrico , Montanaro Lucio , Samorí Bruno
Published: November 2003

Fibronectin (Fn) is an important mediator of bacterial invasions and of persistent infections like that of
Staphylococcus epidermis
. Similar to many other types of cell-protein adhesion, the binding between Fn and
S. epidermidis
takes place under physiological shear rates. We investigated the dynamics of the interaction between individual living
S. epidermidis
cells and single Fn molecules under mechanical force by using the scanning force microscope. The mechanical strength of this interaction and the binding site in the Fn molecule were determined. The energy landscape of the binding/unbinding process was mapped, and the force spectrum and the association and dissociation rate constants of the binding pair were measured. The interaction between
S. epidermidis
cells and Fn molecules is compared with those of two other protein/ligand pairs known to mediate different dynamic states of adhesion of cells under a hydrodynamic flow: the firm adhesion mediated by biotin/avidin interactions, and the rolling adhesion, mediated by L-selectin/P-selectin glycoprotein ligand-1 interactions. The inner barrier in the energy landscape of the Fn case characterizes a high-energy binding mode that can sustain larger deformations and for significantly longer times than the correspondent high-strength L-selectin/P-selectin glycoprotein ligand-1 binding mode. The association kinetics of the former interaction is much slower to settle than the latter. On this basis, the observations made at the macroscopic scale by other authors of a strong lability of the bacterial adhesions mediated by Fn under high turbulent flow are rationalized at the molecular level.
Abnormal osmotic regulation in

trpv4
-/-

mice
Authors: Liedtke Wolfgang , Friedman Jeffrey M.
Published: November 2003

Osmotic homeostasis is one of the most aggressively defended physiological parameters in vertebrates. However, the molecular mechanisms underlying osmotic regulation are poorly understood. The transient receptor potential channel, vanilloid subfamily (TRPV4), is an osmotically activated ion channel that is expressed in circumventricular organs in the mammalian CNS, which is an important site of osmotic sensing. We have generated
trpv4
-null mice and observed abnormalities of their osmotic regulation.
trpv4
-/-
mice drank less water and became more hyperosmolar than did wild-type littermates, a finding that was seen with and without administration of hypertonic saline. In addition, plasma levels of antidiuretic hormone were significantly lower in
trpv4
-/-
mice than in wild-type littermates after a hyperosmotic challenge. Continuous s.c. infusion of the antidiuretic hormone analogue, dDAVP, resulted in systemic hypotonicity in
trpv4
-/-
mice, despite the fact that their renal water reabsorption capacity was normal. Thus, the response to both hyper- and hypoosmolar stimuli is impaired in
trpv4
-/-
mice. After a hyperosmolar challenge, there was markedly reduced expression of c-FOS in the circumventricular organ, the organum vasculosum of the lamina terminalis, of
trpv4
-/-
mice compared with wild-type mice. This finding suggests that there is an impairment of osmotic sensing in the CNS of
trpv4
-/-
mice. These data indicate that TRPV4 is necessary for the normal response to changes in osmotic pressure and functions as an osmotic sensor in the CNS.
Reevaluation of human cytomegalovirus coding potential
Authors: Murphy Eain , Rigoutsos Isidore , Shibuya Tetsuo , Shenk Thomas E.
Published: November 2003

The Bio-Dictionary-based Gene Finder was used to reassess the coding potential of the AD169 laboratory strain of human cytomegalovirus and sequences in the Toledo strain that are missing in the laboratory strain of the virus. The gene-finder algorithm assesses the potential of an ORF to encode a protein based on matches to a database of amino acid patterns derived from a large collection of proteins. The algorithm was used to score all human cytomegalovirus ORFs with the potential to encode polypeptides ≥50 aa in length. As a further test for functionality, the genomes of the chimpanzee, rhesus, and murine cytomegaloviruses were searched for orthologues of the predicted human cytomegalovirus ORFs. The analysis indicates that 37 previously annotated ORFs ought to be discarded, and at least nine previously unrecognized ORFs with relatively strong coding potential should be added. Thus, the human cytomegalovirus genome appears to contain ≈192 unique ORFs with the potential to encode a protein. Support for several of the predictions of ourin silicoanalysis was obtained by sequencing several domains within a clinical isolate of human cytomegalovirus.
Extensive repetitive DNA facilitates prokaryotic genome plasticity
Authors: Aras Rahul A. , Kang Josephine , Tschumi Ariane I. , Harasaki Yasuaki , Blaser Martin J.
Published: November 2003

Prokaryotic genomes are substantially diverse, even when from closely related species, with the resulting phenotypic diversity representing a repertoire of adaptations to specific constraints. Within the microbial population, genome content may not be fixed, as changing selective forces favor particular phenotypes; however, organisms well adapted to particular niches may have evolved mechanisms to facilitate such plasticity. The highly diverse
Helicobacter pylori
is a model for studying genome plasticity in the colonization of individual hosts. For
H. pylori
, neither point mutation, nor intergenic recombination requiring the presence of multiple colonizing strains, is sufficient to fully explain the observed diversity. Here we demonstrate that
H. pylori
has extensive, nonrandomly distributed repetitive chromosomal sequences, and that recombination between identical repeats contributes to the variation within individual hosts. That
H. pylori
is representative of prokaryotes, especially those with smaller (<2 megabases) genomes, that have similarly extensive direct repeats, suggests that recombination between such direct DNA repeats is a widely conserved mechanism to promote genome diversification.
Neural correlates of training-related memory improvement in adulthood and aging
Authors: Nyberg Lars , Sandblom Johan , Jones Sari , Neely Anna Stigsdotter , Petersson Karl Magnus , Ingvar Martin , Bäckman Lars
Published: November 2003

Cognitive studies show that both younger and older adults can increase their memory performance after training in using a visuospatial mnemonic, although age-related memory deficits tend to be magnified rather than reduced after training. Little is known about the changes in functional brain activity that accompany training-induced memory enhancement, and whether age-related activity changes are associated with the size of training-related gains. Here, we demonstrate that younger adults show increased activity during memory encoding in occipito-parietal and frontal brain regions after learning the mnemonic. Older adults did not show increased frontal activity, and only those elderly persons who benefited from the mnemonic showed increased occipito-parietal activity. These findings suggest that age-related differences in cognitive reserve capacity may reflect both a frontal processing deficiency and a posterior production deficiency.
Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading
Authors: Rong James X. , Shapiro Mark , Trogan Eugene , Fisher Edward A.
Published: November 2003

Mouse aortic smooth muscle cells (SMCs) were loaded for 72 h with cholesterol by using cholesterol:methyl-β-cyclodextrin complexes, leading to ≈2-fold and ≈10-fold increases in the contents of total cholesterol and cholesteryl ester, respectively. Foam-cell formation was demonstrated by accumulation of intracellular, Oil Red O-stained lipid droplets. Immunostaining showed decreased protein levels of smooth muscle α-actin and α-tropomyosin and increased levels of macrophage markers CD68 and Mac-2 antigen. Quantitative real-time RT-PCR revealed that after cholesterol loading, the expression of SMC-related genes α-actin, α-tropomyosin, myosin heavy chain, and calponin H1 decreased (to 11.5 ± 0.5%, 29.3 ± 1.4%, 23.8 ± 1.4%, and 3.8 ± 0.5% of unloaded cells, respectively;
P
< 0.05 for all), whereas expression of macrophage-related genes CD68, Mac-2, and ABCA1 mRNA increased (to 709 ± 84%, 330 ± 11%, and 207 ± 13% of unloaded cells, respectively;
P
< 0.05 for all), thereby demonstrating that the protein changes were regulated at the mRNA level. Furthermore, these changes were accompanied by a gain in macrophage-like function as assessed by phagocytotic activity. Expression of vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1, known responders to inflammation, were not changed. In conclusion, cholesterol loading of SMC causes phenotypic changes regulated at the mRNA level that result in a transdifferentiation to a macrophage-like state. This finding suggests that not all foam cells in lesions may have a macrophage origin, despite what is indicated by immunostaining for macrophage-related markers. Furthermore, inflammatory changes in foam cells observed
in vivo
may not be simple consequences of cholesterol accumulation.
Histone sumoylation is associated with transcriptional repression
Authors: Shiio Yuzuru , Eisenman Robert N.
Published: November 2003

Histone proteins are subject to modifications, such as acetylation, methylation, phosphorylation, ubiquitination, glycosylation, and ADP ribosylation, some of which are known to play important roles in the regulation of chromatin structure and function. Here we report that histone H4 is modified by small ubiquitin-related modifier (SUMO) family proteins both
in vivo
and
in vitro
. H4 binds to the SUMO-conjugating enzyme (E2), UBC9, and can be sumoylated in an E1 (SUMO-activating enzyme)- and E2-dependent manner. We present evidence suggesting that histone sumoylation mediates gene silencing through recruitment of histone deacetylase and heterochromatin protein 1.
Discovery of a bacterium, with distinctive dioxygenase, that is responsible for
in situ
biodegradation in contaminated sediment
Authors: Jeon C. O. , Park W. , Padmanabhan P. , DeRito C. , Snape J. R. , Madsen E. L.
Published: November 2003

Microorganisms maintain the biosphere by catalyzing biogeochemical processes, including biodegradation of organic chemical pollutants. Yet seldom have the responsible agents and their respective genes been identified. Here we used field-based stable isotopic probing (SIP) to discover a group of bacteria responsible for
in situ
metabolism of an environmental pollutant, naphthalene. We released
13
C-labeled naphthalene in a contaminated study site to trace the flow of pollutant carbon into the naturally occurring microbial community. Using GC/MS, molecular biology, and classical microbiological techniques we documented
13
CO
2
evolution (2.3% of the dose in 8 h), created a library of 16S rRNA gene clones from
13
C labeled sediment DNA, identified a taxonomic cluster (92 of 95 clones) from the microbial community involved in metabolism of the added naphthalene, and isolated a previously undescribed bacterium (strain CJ2) from site sediment whose 16S rRNA gene matched that of the dominant member (48%) of the clone library. Strain CJ2 is a β proteobacterium closely related to
Polaromonas vacuolata
. Moreover, strain CJ2 hosts the sequence of a naphthalene dioxygenase gene, prevalent in site sediment, detected before only in environmental DNA. This investigative strategy may have general application for elucidating the bases of many biogeochemical processes, hence for advancing knowledge and management of ecological and industrial systems that rely on microorganisms.
Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation
Authors: Akagi Tsuyoshi , Sasai Ken , Hanafusa Hidesaburo
Published: November 2003

Human cells are known to be more refractory than rodent cells against oncogenic transformation
in vitro
. To date, the molecular mechanisms underlying such resistance remain largely unknown. The combination of simian virus 40 early region and H-Ras V12 has been effective for transformation of rat embryo fibroblasts, but not for human cells. However, the additional ectopic expression of the telomerase catalytic subunit (hTERT) was reported to be capable of causing transformation of normal human cells. In this study, however, we demonstrate that the combined expression of the above-mentioned three genetic elements is not always sufficient to transform normal human diploid fibroblasts (HDF). Although the expression and function of these introduced genetic elements were essentially the same, among four HDF, TIG-1 and TIG-3 were resistant to transformation. The other two (BJ and IMR-90) showed transformed phenotypes, but they were much restricted compared with rat embryo fibroblasts in expressing simian virus 40 early region and H-Ras V12. In correlation with these phenotypes, TIG-1 and TIG-3 remained diploid after the introduction of these genetic elements, whereas BJ and IMR-90 became highly aneuploid. These results strongly suggest that the lack of telomerase is not the sole reason for the refractory nature of HDF against transformation and that normal human cells have still undefined intrinsic mechanisms rendering them resistant to oncogenic transformation.
N-desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates N-methyl-
d
-aspartate receptor activity
Authors: Sur Cyrille , Mallorga Pierre J. , Wittmann Marion , Jacobson Marlene A. , Pascarella Danette , Williams Jacinta B. , Brandish Philip E. , Pettibone Douglas J. , Scolnick Edward M. , Conn P. Jeffrey
Published: November 2003

The molecular and neuronal substrates conferring on clozapine its unique and superior efficacy in the treatment of schizophrenia remain elusive. The interaction of clozapine with many G protein-coupled receptors is well documented but less is known about its biologically active metabolite,
N
-desmethylclozapine. Recent clinical and preclinical evidences of the antipsychotic activity of the muscarinic agonist xanomeline prompted us to investigate the effects of
N
-desmethylclozapine on cloned human M1-M5 muscarinic receptors.
N
-desmethylclozapine preferentially bound to M1 muscarinic receptors with an IC
50
of 55 nM and was a more potent partial agonist (EC
50
, 115 nM and 50% of acetylcholine response) at this receptor than clozapine. Furthermore, pharmacological and site-directed mutagenesis studies suggested that
N
-desmethylclozapine preferentially activated M1 receptors by interacting with a site that does not fully overlap with the acetylcholine orthosteric site. As hypofunction of
N
-methyl-
d
-aspartate (NMDA) receptor-driven neuronal ensembles has been implicated in psychotic disorders, the neuronal activity of
N
-desmethylclozapine was electrophysiologically investigated in hippocampal rat brain slices.
N
-desmethylclozapine was shown to dose-dependently potentiate NMDA receptor currents in CA1 pyramidal cells by 53% at 100 nM, an effect largely mediated by activation of muscarinic receptors. Altogether, our observations provide direct evidence that the brain penetrant metabolite
N
-desmethylclozapine is a potent, allosteric agonist at human M1 receptors and is able to potentiate hippocampal NMDA receptor currents through M1 receptor activation. These observations raise the possibility that
N
-desmethylclozapine contributes to clozapine's clinical activity in schizophrenics through modulation of both muscarinic and glutamatergic neurotransmission.
Quantitative trait symmetry independent of Hsp90 buffering: Distinct modes of genetic canalization and developmental stability
Authors: Milton Claire C. , Huynh Brandon , Batterham Philip , Rutherford Suzanne L. , Hoffmann Ary A.
Published: November 2003

The Hsp90 chaperone buffers development against a wide range of morphological changes in many organisms and in
Drosophila
masks the effects of hidden genetic variation. Theory predicts that genetic and nongenetic buffering will share common mechanisms. For example, it is argued that Hsp90 genetic buffering evolved solely as a by-product of environmental buffering, and that Hsp90 should mask morphological deviations from any source. To test this idea, we examined the effect of Hsp90 on purely nongenetic variation in phenotype, measured as differences between the left and right sides of several bilaterally symmetrical bristle and wing traits in individual flies. Consistent with previous reports, Hsp90 buffered the expression of rare morphogenic variants specific to particular genetic backgrounds. However, neither trait-by-trait nor global asymmetry was affected in outbred flies treated with an Hsp90 inhibitor or across a series of inbred genetic backgrounds from a wild population tested in isogenic F
1
heterozygotes carrying either (
i
) a dominant negative Hsp90 allele on a mutant 3rd chromosome or (
ii
) a null
P
-insertion mutation, which was introgressed into the control genetic background on all chromosomes. By contrast, Hsp90-regulated trait means and significant effects of sex, temperature, and genetic background on trait symmetry were clearly detected. We conclude that, by maintaining the function of signaling proteins, Hsp90 masks variation affecting target pathways and traits in populations independent of purely nongenetic sources of variation, refuting the idea that a single Hsp90-dependent process generally controls genetic canalization and developmental stability.
Solution structure of ade novoprotein from a designed combinatorial library
Authors: Wei Yinan , Kim Seho , Fela David , Baum Jean , Hecht Michael H.
Published: November 2003

Combinatorial libraries ofde novoamino acid sequences can provide a rich source of diversity for the discovery of novel proteins. Randomly generated sequences, however, rarely fold into well ordered protein-like structures. To enhance the quality of a library, diversity must be focused into those regions of sequence space most likely to yield well folded structures. We have constructed focused libraries ofde novosequences by designing the binary pattern of polar and nonpolar amino acids to favor structures that contain abundant secondary structure, while simultaneously burying hydrophobic side chains in the protein interior and exposing hydrophilic side chains to solvent. Because binary patterning specifies only the polar/nonpolar periodicity, but not the identities of the side chains, detailed structural features, including packing interactions, cannot be designeda priori. Can binary patterned libraries nonetheless encode well folded proteins? An unambiguous answer to this question requires determination of a 3D structure. We used NMR spectroscopy to determine the structure of S-824, a novel protein from a recently constructed library of 102-residue sequences. This library is “naïve” in that it has not been subjected to high-throughput screens or directed evolution. The experimentally determined structure of S-824 is a four-helix bundle, as specified by the design. As dictated by the binary-code strategy, nonpolar side chains are buried in the protein interior, and polar side chains are exposed to solvent. The polypeptide backbone and buried side chains are well ordered, demonstrating that S-824 isnota molten globule and forms a unique structure. These results show that amino acid sequences that have neither been selected by evolution, nor designed by computer, nor isolated by high-throughput screening, can form native-like structures. These findings validate the binary-code strategy as an effective method for producing vast collections of well foldedde novoproteins.
Convergent neofunctionalization by positive Darwinian selection after ancient recurrent duplications of the
xanthine dehydrogenase
gene
Authors: Rodríguez-Trelles Francisco , Tarrío Rosa , Ayala Francisco J.
Published: November 2003

Gene duplication is a primary source of molecular substrate for the emergence of evolutionary novelties. The chances for redundant gene sequences to evolve new functions are small compared with the probability that the copies become disabled by deleterious mutations. Functional divergence after gene duplication can result in two alternative evolutionary fates: one copy acquires a novel function (neofunctionalization), or each copy adopts part of the tasks of their parental gene (subfunctionalization). The relative prevalence of each outcome is unknown. Similarly unknown is the relative importance of positive selection versus random fixation of neutral mutations. Aldehyde oxidase (
Ao
) and xanthine dehydrogenase (
Xdh
) genes encode two complex members of the xanthine oxidase family of molybdo-flavoenzymes that carry different functions.
Ao
is known to have originated from a duplicate of an
Xdh
gene in eukaryotes, before the origin of multicellularity. We show that (
i
)
Ao
evolved independently twice from two different
Xdh
paralogs, the second time in the chordates, before the diversification of vertebrates; (
ii
) after each duplication, the
Ao
duplicate underwent a period of rapid evolution during which identical sites across the two molecules, involving the flavin adenine dinucleotide and substrate-binding pockets, were subjected to intense positive Darwinian selection; and (
iii
) the second
Ao
gene likely endured two periods of redundancy, initially as a duplicate of
Xdh
and later as a functional equivalent of the old
Ao
, which is currently absent from the vertebrate genome. Caution is appropriate in structural genomics when using sequence similarity for assigning protein function.
Axonal transport of eukaryotic translation elongation factor 1α mRNA couples transcription in the nucleus to long-term facilitation at the synapse
Authors: Giustetto Maurizio , Hegde Ashok N. , Si Kausik , Casadio Andrea , Inokuchi Kaoru , Pei Wanzheng , Kandel Eric R. , Schwartz James H.
Published: November 2003

Long-term synaptic plasticity requires both gene expression in the nucleus and local protein synthesis at synapses. The effector proteins that link molecular events in the cell body with local maintenance of synaptic strength are not known. We now show that treatment with serotonin (5-HT) that produces long-term facilitation induces theAplysiaeukaryotic translation elongation factor 1α (Ap-eEF1A) as a late gene that might serve this coupling function in sensory neurons. Although the translation factor is induced, it is not transported into axon processes when the stimulation with 5-HT was restricted to the cell body. In contrast, its mRNA is transported when 5-HT was applied to both cell body and synapses. Intracellular injection of antisense oligonucleotides or antibodies that block the induction and expression of Ap-eEF1A do not affect the initial expression of long-term facilitation but do block its maintenance beyond 24 h. The transport of eEF1A protein and its mRNA to nerve terminals suggests that the translation factor plays a role in the local protein synthesis that is essential for maintaining newly formed synapses.
The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor
Authors: Yang Haitao , Yang Maojun , Ding Yi , Liu Yiwei , Lou Zhiyong , Zhou Zhe , Sun Lei , Mo Lijuan , Ye Sheng , Pang Hai , Gao George F. , Anand Kanchan , Bartlam Mark , Hilgenfeld Rolf , Rao Zihe
Published: November 2003

A newly identified severe acute respiratory syndrome coronavirus (SARS-CoV), is the etiological agent responsible for the outbreak of SARS. The SARS-CoV main protease, which is a 33.8-kDa protease (also called the 3C-like protease), plays a pivotal role in mediating viral replication and transcription functions through extensive proteolytic processing of two replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Here, we report the crystal structures of the SARS-CoV main protease at different pH values and in complex with a specific inhibitor. The protease structure has a fold that can be described as an augmented serine-protease, but with a Cys-His at the active site. This series of crystal structures, which is the first, to our knowledge, of any protein from the SARS virus, reveal substantial pH-dependent conformational changes, and an unexpected mode of inhibitor binding, providing a structural basis for rational drug design.
Endogenous neosynthesis vs. cross-presentation of viral antigens for cytotoxic T cell priming
Authors: Freigang Stefan , Egger Denise , Bienz Kurt , Hengartner Hans , Zinkernagel Rolf M.
Published: November 2003

Induction of antiviral cytotoxic T lymphocytes (CTLs) has been proposed to require cross-presentation of viral antigens derived from infected extralymphatic host cells by antigen-presenting cells (APC). This postulated mechanism of cross-priming is thought to be essential for CTL responses against viruses that do not infect professional APC, e.g., because of absence of the specific virus receptor. Here, we show for the human pathogen poliovirus that naturally nonpermissive murine APC acquire viral RNAin vivoindependently of the cellular virus receptor. Uptake of poliovirus or polioviral RNA initiated neosynthesis of viral antigen to an extent sufficient to prime CTLsin vivo, which were detectable 2-3 wk after infection. Our results do not only indicate that experiments studying cross-presentation and cross-priming by using potentially amplifiable or translatable materials need careful examination, but they also question the general biological importance of cross-presentation and cross-priming in antiviral CTL responses.
Allosteric modulation of group III metabotropic glutamate receptor 4: A potential approach to Parkinson's disease treatment
Authors: Marino Michael J. , Williams David L. , O'Brien Julie A. , Valenti Ornella , McDonald Terrence P. , Clements Michelle K. , Wang Ruiping , DiLella Anthony G. , Hess J. Fred , Kinney Gene G. , Conn P. Jeffrey
Published: November 2003

Parkinson's disease (PD) is a debilitating movement disorder that afflicts >1 million people in North America. Current treatments focused on dopamine-replacement strategies ultimately fail in most patients because of loss of efficacy and severe adverse effects that worsen as the disease progresses. The recent success of surgical approaches suggests that a pharmacological intervention that bypasses the dopamine system and restores balance in the basal ganglia motor circuit may provide an effective treatment strategy. We previously identified the metabotropic glutamate receptor 4 (mGluR4) as a potential drug target and predicted that selective activation of mGluR4 could provide palliative benefit in PD. We now report that
N
-phenyl-7-(hydroxylimino)cyclopropa[
b
]chromen-1a-carboxamide (PHCCC) is a selective allosteric potentiator of mGluR4. This compound selectively potentiated agonist-induced mGluR4 activity in cultured cells expressing this receptor and did not itself act as an agonist. Furthermore, PHCCC potentiated the effect of
l
-(+)-2-amino-4-phosphonobutyric acid in inhibiting transmission at the striatopallidal synapse. Modulation of the striatopallidal synapse has been proposed as a potential therapeutic target for PD, in that it may restore balance in the basal ganglia motor circuit. Consistent with this, PHCCC produced a marked reversal of reserpine-induced akinesia in rats. The closely related analogue 7-(hydroxylimino)cyclopropachromen-1a-carboxamide ethyl ester, which does not potentiate mGluR4, had no effect in this model. These results are evidence for
in vivo
behavioral effects of an allosteric potentiator of mGluRs and suggest that potentiation of mGluR4 may be a useful therapeutic approach to the treatment of PD.
The
Drosophila
synaptotagmin-like protein bitesize is required for growth and has mRNA localization sequences within its open reading frame
Authors: Serano Julia , Rubin Gerald M.
Published: November 2003

The vertebrate synaptotagmin-like protein granuphilin binds to the vesicle-trafficking proteins Rab27a and Munc18 and can modulate exocytosis of insulin-containing secretory granules in pancreatic beta cell lines. Here, we report the molecular and genetic characterization of bitesize, a granuphilin homolog and the only
Drosophila
synaptotagmin-like protein. Mutations that affect bitesize have reduced cell size and number, resulting in smaller animals that develop slowly. We also show that at least two classes of
bitesize
transcripts are localized to the apical plasma membrane in polarized epithelial cells. Whereas most cis-acting mRNA localization sequences map to 3′ untranslated regions,
bitesize
contains a 2.2-kb sequence within its ORF that is necessary and sufficient for apical localization. Thus, we have found that
bitesize
is a metazoan example of a transcript for which all identifiable mRNA localization sequences are contained within the protein-coding region.
Invasion, competitive dominance, and resource use by exotic and native California grassland species
Authors: Seabloom Eric W. , Harpole W. Stanley , Reichman O. J. , Tilman David
Published: November 2003

The dynamics of invasive species may depend on their abilities to compete for resources and exploit disturbances relative to the abilities of native species. We test this hypothesis and explore its implications for the restoration of native ecosystems in one of the most dramatic ecological invasions worldwide, the replacement of native perennial grasses by exotic annual grasses and forbs in 9.2 million hectares of California grasslands. The long-term persistence of these exotic annuals has been thought to imply that the exotics are superior competitors. However, seed-addition experiments in a southern California grassland revealed that native perennial species, which had lower requirements for deep soil water, soil nitrate, and light, were strong competitors, and they markedly depressed the abundance and fecundity of exotic annuals after overcoming recruitment limitations. Native species reinvaded exotic grasslands across experimentally imposed nitrogen, water, and disturbance gradients. Thus, exotic annuals are not superior competitors but rather may dominate because of prior disturbance and the low dispersal abilities and extreme current rarity of native perennials. If our results prove to be general, it may be feasible to restore native California grassland flora to at least parts of its former range.
Rescue of mutants of the tumor suppressor p53 in cancer cells by a designed peptide
Authors: Issaeva Natalia , Friedler Assaf , Bozko Przemyslaw , Wiman Klas G. , Fersht Alan R. , Selivanova Galina
Published: November 2003

We designed a series of nine-residue peptides that bound to a defined site on the tumor suppressor p53 and stabilized it against denaturation. To test whether the peptides could act as chaperones and rescue the tumor-suppressing function of oncogenic mutants of p53 in living cells, we treated human tumor cells with the fluorescein-labeled peptide Fl-CDB3 (fluorescent derivative of CDB3). Before treatment, the mutant p53 in the cell was predominantly denatured. Fl-CDB3 was taken up into the cytoplasm and nucleus and induced a substantial up-regulation of wild-type p53 protein and representative mutants. The mutants, His-273 and His-175 p53, adopted the active conformation, with a dramatic decrease in the fraction of denatured protein. In all cases, there was p53-dependent induction of expression of the p53 target genes
mdm2, gadd45
, and
p21
, accompanied by p53-dependent partial restoration of apoptosis. Fl-CDB3 sensitized cancer cells that carried wild-type p53 to p53-dependent γ-radiation-induced apoptosis. Although Fl-CDB3 did not elicit a full biological response, it did bind to and rescue p53 in cells and so can serve as a lead for the development of novel drugs for anticancer therapy.
Unifying features in protein-folding mechanisms
Authors: Gianni Stefano , Guydosh Nicholas R. , Khan Faaizah , Caldas Teresa D. , Mayor Ugo , White George W. N. , DeMarco Mari L. , Daggett Valerie , Fersht Alan R.
Published: November 2003

We compare the folding of representative members of a protein superfamily by experiment and simulation to investigate common features in folding mechanisms. The homeodomain superfamily of three-helical, single-domain proteins exhibits a spectrum of folding processes that spans the complete transition from concurrent secondary and tertiary structure formation (nucleation-condensation mechanism) to sequential secondary and tertiary formation (framework mechanism). The unifying factor in their mechanisms is that the transition state for (un)folding is expanded and very native-like, with the proportion and degree of formation of secondary and tertiary interactions varying. There is a transition, or slide, from the framework to nucleation-condensation mechanism with decreasing stability of the secondary structure. Thus, framework and nucleation-condensation are different manifestations of an underlying common mechanism.
Identification and characterization of a cyclooxygenase-like enzyme fromEntamoeba histolytica
Authors: Dey Indranil , Keller Kathy , Belley Adam , Chadee Kris
Published: November 2003

The intestinal protozoan parasiteEntamoeba histolyticaremains a significant cause of morbidity and mortality worldwide. However, almost nothing is known about the molecules secreted by the parasite that modulate host immune responses or epithelial barrier function in the colon. Herein, we describe the isolation and characterization of a cyclooxygenase (COX)-like enzyme inE. histolyticathat is responsible for the biosynthesis of prostaglandin (PG)E2. PGE2produced by ameba was constitutive but highly dependent on exogenous arachidonic acid substrate. COX-like activity and the immunoreactive protein were localized to the nuclear fraction ofE. histolytica. The COX-like protein (72 kDa) was microsequenced and cloned by reverse transcriptase PCR. Ameba COX showed little homology with COX-1/2 enzymes from different species at the nucleotide and amino acid levels. Surprisingly, the arachidonate-binding domain and heme-coordinating and catalytic sites, which are conserved in other species, were absent in ameba. Ameba COX expressed inEscherichia colidemonstrated COX-like enzyme activityin vitroby converting arachidonic acid into PGE2but not into PGD2or PGF2α. COX activity was inhibited with 1 mM aspirin but not with indomethacin or COX-1/2-specific inhibitors. Taken together, these studies reveal thatE. histolyticaproduces PGE2, by means of a previously undescribed ancestral COX-like enzyme, which could play a major role in pathogenesis and immune evasion.
Adaptive evolution in the
Arabidopsis
MADS-box gene family inferred from its complete resolved phylogeny
Authors: Martínez-Castilla León Patricio , Alvarez-Buylla Elena R.
Published: November 2003

Gene duplication is a substrate of evolution. However, the relative importance of positive selection versus relaxation of constraints in the functional divergence of gene copies is still under debate. Plant MADS-box genes encode transcriptional regulators key in various aspects of development and have undergone extensive duplications to form a large family. We recovered 104 MADS sequences from the
Arabidopsis
genome. Bayesian phylogenetic trees recover type II lineage as a monophyletic group and resolve a branching sequence of monophyletic groups within this lineage. The type I lineage is comprised of several divergent groups. However, contrasting gene structure and patterns of chromosomal distribution between type I and II sequences suggest that they had different evolutionary histories and support the placement of the root of the gene family between these two groups. Site-specific and site-branch analyses of positive Darwinian selection (PDS) suggest that different selection regimes could have affected the evolution of these lineages. We found evidence for PDS along the branch leading to flowering time genes that have a direct impact on plant fitness. Sites with high probabilities of having been under PDS were found in the MADS and K domains, suggesting that these played important roles in the acquisition of novel functions during MADS-box diversification. Detected sites are targets for further experimental analyses. We argue that adaptive changes in MADS-domain protein sequences have been important for their functional divergence, suggesting that changes within coding regions of transcriptional regulators have influenced phenotypic evolution of plants.
The inhibitory potential of Fc receptor homolog 4 on memory B cells
Authors: Ehrhardt Götz R. A. , Davis Randall S. , Hsu Joyce T. , Leu Chuen-Miin , Ehrhardt Annette , Cooper Max D.
Published: November 2003

Fc receptor homolog 4 (FcRH4) is a B cell-specific member of the recently identified family of FcRHs whose intracellular domain contains three potential immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The signaling potential of this receptor, shown here to be preferentially expressed by memory B cells, was compared with the inhibitory receptor FcγRIIb in B cells expressing either WT FcγRIIb or chimeric proteins in which the intracellular domain of FcRH4 was fused to the transmembrane and extracellular domains of FcγRIIb. Coligation of the FcγRIIb/FcRH4 chimeric protein with the B cell receptor (BCR) led to tyrosine phosphorylation of the two membrane-distal tyrosines and profound inhibition of BCR-mediated calcium mobilization, whole cell tyrosine phosphorylation, and mitogen-activated protein (MAP)-kinase activation. Mutational analysis of the FcRH4 cytoplasmic region indicated that the two membrane-distal ITIMs are essential for this inhibitory potential. Phosphopeptides corresponding to these ITIMs could bind the Src homology 2 (SH2) domain-containing tyrosine phosphatases SHP-1 and SHP-2, which associated with the WT FcRH4 and with mutants having inhibitory capability. These findings indicate the potential for FcRH4 to abort B cell receptor signaling by recruiting SHP-1 and SHP-2 to its two membrane distal ITIMs.
A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells
Authors: Wolfsgruber Wiebke , Feil Susanne , Brummer Sabine , Kuppinger Oliver , Hofmann Franz , Feil Robert
Published: November 2003

Nitric oxide (NO) exerts both antiatherogenic and proatherogenic effects, but the cellular and molecular mechanisms that contribute to modulation of atherosclerosis by NO are not understood completely. The cGMP-dependent protein kinase I (cGKI) is a potential mediator of NO signaling in vascular smooth muscle cells (SMCs). Postnatal ablation of cGKI selectively in the SMCs of mice reduced atherosclerotic lesion area, demonstrating that smooth muscle cGKI promotes atherogenesis. Cell-fate mapping indicated that cGKI is involved in the development of SMC-derived plaque cells. Activation of endogenous cGKI in primary aortic SMCs resulted in cells with increased levels of proliferation; increased levels of vascular cell adhesion molecule-1, peroxisome proliferator-activated receptor γ, and phosphatidylinositol 3-kinase/Akt signaling; and decreased plasminogen activator inhibitor 1 mRNA, which all are potentially proatherogenic properties. Taken together, these results highlight the pathophysiologic significance of vascular SMCs in atherogenesis and identify a key role for cGKI in the development of atherogenic SMCs
in vitro
and
in vivo
. We suggest that activation of smooth muscle cGKI contributes to the proatherogenic effect of NO and that inhibition of cGKI might be a therapeutic option for treating atherosclerosis in humans.
Estimating mean lifetime
Authors: Bongaarts John , Feeney Griffith
Published: November 2003

The life expectancy implied by current age-specific mortality rates is calculated with life table methods that are among the oldest and most fundamental tools of demography. We demonstrate that these conventional estimates of period life expectancy are affected by an undesirable “tempo effect.” The tempo effect is positive when the mean age at death is rising and negative when the mean is declining. Estimates of the effect for females in three countries with high and rising life expectancy range from 1.6 yr in the U.S. and Sweden to 2.4 yr in France for the period 1980–1995.
An open conformation of mammalian cytochrome P450 2B4 at 1.6-Å resolution
Authors: Scott Emily E. , He You Ai , Wester Michael R. , White Mark A. , Chin Christopher C. , Halpert James R. , Johnson Eric F. , Stout C. David
Published: November 2003

The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-Å crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the α-helical and β-sheet domains without perturbing the overall P450 fold. This cleft is primarily formed by helices B′ to C and F to G. The conformation of these regions is dramatically different from that of the other structurally defined mammalian P450, 2C5/3LVdH, in which the F to G and B′ to C regions encapsulate one side of the active site to produce a closed form of the enzyme. The open conformation of 2B4 is trapped by reversible formation of a homodimer in which the residues between helices F and G of one molecule partially fill the open cleft of a symmetry-related molecule, and an intermolecular coordinate bond occurs between H226 and the heme iron. This dimer is observed both in solution and in the crystal. Differences between the structures of 2C5 and 2B4 suggest that defined regions of xenobiotic metabolizing P450s may adopt a substantial range of energetically accessible conformations without perturbing the overall fold. This conformational flexibility is likely to facilitate substrate access, metabolic versatility, and product egress.
Contribution of climate-driven change in continental water storage to recent sea-level rise
Authors: Milly P. C. D. , Cazenave A. , Gennero C.
Published: November 2003

Using a global model of continental water balance, forced by interannual variations in precipitation and near-surface atmospheric temperature for the period 1981–1998, we estimate the sea-level changes associated with climate-driven changes in storage of water as snowpack, soil water, and ground water; storage in ice sheets and large lakes is not considered. The 1981–1998 trend is estimated to be 0.12 mm/yr, and substantial interannual fluctuations are inferred; for 1993–1998, the trend is 0.25 mm/yr. At the decadal time scale, the terrestrial contribution to eustatic (i.e., induced by mass exchange) sea-level rise is significantly smaller than the estimated steric (i.e., induced by density changes) trend for the same period, but is not negligibly small. In the model the sea-level rise is driven mainly by a downtrend in continental precipitation during the study period, which we believe was generated by natural variability in the climate system.
Boundary of quantum evolution under decoherence
Authors: Khaneja Navin , Luy Burkhard , Glaser Steffen J.
Published: November 2003

Relaxation effects impose fundamental limitations on our ability to coherently control quantum mechanical phenomena. In this article, we use principles of optimal control theory to establish physical limits on how closely a quantum mechanical system can be steered to a desired target state in the presence of relaxation. In particular, we explicitly compute the maximum amplitude of coherence or polarization that can be transferred between coupled heteronuclear spins in large molecules at high magnetic fields in the presence of relaxation. Very general decoherence mechanisms that include cross-correlated relaxation have been included in our analysis. We give analytical characterization for the pulse sequences (control laws) that achieve these physical limits and provide supporting experimental evidence. Exploitation of cross-correlation effects has recently led to the development of powerful methods in NMR spectroscopy to study very large biomolecules in solution. For two heteronuclear spins, we demonstrate with experiments that cross-correlated relaxation optimized pulse (CROP) sequences provide significant gains over the state-of-the-art methods. It is shown that despite large relaxation rates, coherence can be transferred between coupled spins without any loss in special cases where cross-correlated relaxation rates can be tuned to autocorrelated relaxation rates.
Identification of functional similarities between proteins using directed evolution
Authors: Christ Daniel , Winter Greg
Published: November 2003

Protein sequences are often highly redundant and evolution can change them beyond recognition. It can therefore be difficult to identify proteins with functional or structural similarities by inspection of their sequences. Here we have used an experimental evolutionary approach to detect hidden similarities between the antisense RNA-binding protein Rop and other proteins. We created an envelope of functional Rop mutants by combinatorial mutagenesis, used the compilation of mutant sequences to search a database of protein structures, and thereby identified a segment of the enzyme valyl-tRNA-synthetase (ValRS). Further inspection revealed that the structures of the RNA-binding sites of both proteins are highly related, as indeed are the RNA ligands. From the known 3D structure of the ValRS in complex with tRNA, we were able to build a model of an RNA hairpin pair in complex with Rop that has proved to be consistent with the biochemical and NMR data for the interaction between Rop and RNA hairpins. We suggest that this approach (mutational envelope scanning), by generating sequence information
de novo
, can help uncover hidden similarities between proteins.
The two rotor components of yeast mitochondrial ATP synthase are mechanically coupled by subunit δ
Authors: Duvezin-Caubet Stéphane , Caron Matthieu , Giraud Marie-France , Velours Jean , di Rago Jean-Paul
Published: November 2003

The mitochondrial ATP synthase is made of a membrane-integrated F
0
component that forms a proton-permeable pore through the inner membrane and a globular peripheral F
1
domain where ATP is synthesized. The catalytic mechanism is thought to involve the rotation of a 10-12
c
subunit ring in the F
0
together with the γ subunit of F
1
. An important and not yet resolved question is to define precisely how the γ subunit is connected with the
c
-ring. In this study, using a doxycycline-regulatable expression system, we provide direct evidence that the rest of the enzyme can assemble without the δ subunit of F
1
, and we show that δ-less mitochondria are uncoupled because of an F
0
-mediated proton leak. Based on these observations, and taking into account high-resolution structural models, we propose that subunit δ plays a key role in the mechanical coupling of the
c
-ring to subunit γ.