bluuebunny/crossref_metadata_2025 huggingface dataset:
Distributional regimes for the number ofk-word matches between two random sequences
Authors: Lippert Ross A. , Huang Haiyan , Waterman Michael S.
Published: October 2002

When comparing two sequences, a natural approach is to count the number ofk-letter words the two sequences have in common. No positional information is used in the count, but it has the virtue that the comparison time is linear with sequence length. For this reason this statisticD2and certain transformations ofD2are used for EST sequence database searches. In this paper we begin the rigorous study of the statistical distribution ofD2. Using an independence model of DNA sequences, we derive limiting distributions by means of the Stein and Chen–Stein methods and identify three asymptotic regimes, including compound Poisson and normal. The compound Poisson distribution arises when the word sizekis large and word matches are rare. The normal distribution arises when the word size is small and matches are common. Explicit expressions for what is meant by large and small word sizes are given in the paper. However, when word size is small and the letters are uniformly distributed, the anticipated limiting normal distribution does not always occur. In this situation the uniform distribution provides the exception to other letter distributions. Therefore a naive, one distribution fits all, approach toD2statistics could easily create serious errors in estimating significance.
A simple physical model for binding energy hot spots in protein–protein complexes
Authors: Kortemme Tanja , Baker David
Published: October 2002

Protein–protein recognition plays a central role in most biological processes. Although the structures of many protein–protein complexes have been solved in molecular detail, general rules describing affinity and selectivity of protein–protein interactions do not accurately account for the extremely diverse nature of the interfaces. We investigate the extent to which a simple physical model can account for the wide range of experimentally measured free energy changes brought about by alanine mutation at protein–protein interfaces. The model successfully predicts the results of alanine scanning experiments on globular proteins (743 mutations) and 19 protein–protein interfaces (233 mutations) with average unsigned errors of 0.81 kcal/mol and 1.06 kcal/mol, respectively. The results test our understanding of the dominant contributions to the free energy of protein–protein interactions, can guide experiments aimed at the design of protein interaction inhibitors, and provide a stepping-stone to important applications such as interface redesign.
Genomic analysis of uncultured marine viral communities
Authors: Breitbart Mya , Salamon Peter , Andresen Bjarne , Mahaffy Joseph M. , Segall Anca M. , Mead David , Azam Farooq , Rohwer Forest
Published: October 2002

Viruses are the most common biological entities in the oceans by an order of magnitude. However, very little is known about their diversity. Here we report a genomic analysis of two uncultured marine viral communities. Over 65% of the sequences were not significantly similar to previously reported sequences, suggesting that much of the diversity is previously uncharacterized. The most common significant hits among the known sequences were to viruses. The viral hits included sequences from all of the major families of dsDNA tailed phages, as well as some algal viruses. Several independent mathematical models based on the observed number of contigs predicted that the most abundant viral genome comprised 2–3% of the total population in both communities, which was estimated to contain between 374 and 7,114 viral types. Overall, diversity of the viral communities was extremely high. The results also showed that it would be possible to sequence the entire genome of an uncultured marine viral community.
Expanding protein universe and its origin from the biological Big Bang
Authors: Dokholyan Nikolay V. , Shakhnovich Boris , Shakhnovich Eugene I.
Published: October 2002

The bottom-up approach to understanding the evolution of organisms is by studying molecular evolution. With the large number of protein structures identified in the past decades, we have discovered peculiar patterns that nature imprints on protein structural space in the course of evolution. In particular, we have discovered that the universe of protein structures is organized hierarchically into a scale-free network. By understanding the cause of these patterns, we attempt to glance at the very origin of life.
A
Drosophila
dopamine 2-like receptor: Molecular characterization and identification of multiple alternatively spliced variants
Authors: Hearn Mark G. , Ren Yong , McBride Edward W. , Reveillaud Isabelle , Beinborn Martin , Kopin Alan S.
Published: October 2002

Dopamine is an important neurotransmitter in the central nervous system of both
Drosophila
and mammals. Despite the evolutionary distance, functional parallels exist between the fly and mammalian dopaminergic systems, with both playing roles in modulating locomotor activity, sexual function, and the response to drugs of abuse. In mammals, dopamine exerts its effects through either dopamine 1-like (D1-like) or D2-like G protein-coupled receptors. Although pharmacologic data suggest the presence of both receptor subtypes in insects, only cDNAs encoding D1-like proteins have been isolated previously. Here we report the cloning and characterization of a newly discovered
Drosophila
dopamine receptor. Sequence analysis reveals that this putative protein shares highest homology with known mammalian dopamine 2-like receptors. Eight isoforms of the
Drosophila
D2-like receptor (DD2R) transcript have been identified, each the result of alternative splicing. The encoded heptahelical receptors range in size from 461 to 606 aa, with variability in the length and sequence of the third intracellular loop. Pharmacologic assessment of three DD2R isoforms, DD2R-606, DD2R-506, and DD2R-461, revealed that among the endogenous biogenic amines, dopamine is most potent at each receptor. As established for mammalian D2-like receptors, stimulation of the
Drosophila
homologs with dopamine triggers pertussis toxin-sensitive Gi/o-mediated signaling. The D2-like receptor agonist, bromocriptine, has nanomolar potency at DD2R-606, -506, and -461, whereas multiple D2-like receptor antagonists (as established with mammalian receptors) have markedly reduced if any affinity when assessed at the fly receptor isoforms. The isolation of cDNAs encoding
Drosophila
D2-like receptors extends the range of apparent parallels between the dopaminergic system in flies and mammals. Pharmacologic and genetic manipulation of the DD2Rs will provide the opportunity to better define the physiologic role of these proteins
in vivo
and further explore the utility of invertebrates as a model system for understanding dopaminergic function in higher organisms.
Near strongly resonant periodic orbits in a Hamiltonian system
Authors: Gelfreich Vassili
Published: October 2002

We study an analytic Hamiltonian system near a strongly resonant periodic orbit. We introduce a modulus of local analytic classification. We provide asymptotic formulae for the exponentially small splitting of separatrices for bifurcating hyperbolic periodic orbits. These formulae confirm a conjecture formulated by V. I. Arnold in the early 1970s.
Multiple spatiotemporal modes of actin reorganization by NMDA receptors and voltage-gated Ca
2+
channels
Authors: Furuyashiki Tomoyuki , Arakawa Yoshiki , Takemoto-Kimura Sayaka , Bito Haruhiko , Narumiya Shuh
Published: October 2002

Cytoskeleton is believed to contribute to activity-dependent processes underlying neuronal plasticity, such as regulations of cellular morphology and localization of signaling proteins. However, how neuronal activity controls actin cytoskeleton remains obscure. Taking advantage of confocal imaging of enhanced GFP-actin in the primary culture of hippocampal neurons, we show that synaptic activity induces multiple types of actin reorganization, both at the spines and at the somatic periphery. Activation of
N
-methyl-
d
-aspartate receptors, accompanied with a local rise in [Ca
2+
]i, was sufficient to trigger a slow and sustained recruitment of actin into dendritic spines. In contrast, opening of voltage-gated Ca
2+
channels rapidly and reversibly enhanced cortical actin at the somatic periphery but not in the spines, in keeping with a high transient rise in somatic [Ca
2+
]i. These data suggest that spatiotemporal dynamics of [Ca
2+
]i, triggered by activation of
N
-methyl-
d
-aspartate receptors and voltage-gated Ca
2+
channels, provides the molecular basis for activity-dependent actin remodeling.
Stabilities and conformations of Alzheimer's β-amyloid peptide oligomers (Aβ
16
–
22
, Aβ
16–35
, and Aβ
10
–
35
): Sequence effects
Authors: Ma Buyong , Nussinov Ruth
Published: October 2002

Previously, we have studied the minimal oligomer size of an aggregate amyloid seed and the mechanism of seed growth with a multilayer β-sheet model. Under high temperature simulation conditions, our approach can test the stability of possible amyloid forms. Here, we report our study of oligomers of Alzheimer's amyloid β-peptide (Aβ) fragments 16–22, 16–35, and 10–35 (abbreviated Aβ
16–22
, Aβ
16–35
, and Aβ
10–35
, respectively). Our simulations indicate that an antiparallel β-sheet orientation is the most stable for the Aβ
16–22
, in agreement with a solid state NMR-based model [Balbach, J. J., Ishii, Y., Antzutkin, O. N., Leapman, R. D., Rizzo, N. W.,
et al
. (2000)
Biochemistry
39, 13748–13759]. A model with twenty-four Aβ
16–22
strands indicates a highly twisted fibril. Whereas the short Aβ
16–22
and Aβ
24–36
may exist in fully extended form, the linear parallel β-sheets for Aβ
16–35
appear impossible, mainly because of the polar region in the middle of the 16–35 sequence. However, a bent double-layered hairpin-like structure (called
hook
) with the polar region at the turn forms parallel β-sheets with higher stability. An intra-strand salt-bridge (D23-K28) stabilizes the bent hairpin-like hook structure. The bent double-β-sheet model for the Aβ
10–35
similarly offers oligomer stability.
A nucleolar TAR decoy inhibitor of HIV-1 replication
Authors: Michienzi Alessandro , Li Shirley , Zaia John A. , Rossi John J.
Published: October 2002

Tat is a critical regulatory factor in HIV-1 gene expression. It mediates the transactivation of transcription from the HIV-1 LTR by binding to the transactivation response (TAR) element in a complex with cyclin T1. Because of its critical and early role in HIV gene expression, Tat and its interaction with the TAR element constitute important therapeutic targets for the treatment of HIV-1 infection. Based on the known nucleolar localization properties of Tat, we constructed a chimeric small nucleolar RNA-TAR decoy that localizes to the nucleoli of human cells and colocalizes in the nucleolus with a Tat-enhanced GFP fusion protein. When the chimeric RNA was stably expressed in human T lymphoblastoid CEM cells it potently inhibited HIV-1 replication. These results demonstrate that the nucleolar trafficking of Tat is critical for HIV-1 replication and suggests a role for the nucleolus in HIV-1 viral replication.
Biodirected epitaxial nanodeposition of polymers on oriented macromolecular templates
Authors: Kondo Tetsuo , Nojiri Masanobu , Hishikawa Yukako , Togawa Eiji , Romanovicz Dwight , Brown R. Malcolm
Published: October 2002

Biodirected epitaxial nanodeposition of polymers was achieved on a template with an oriented molecular surface.
Acetobacter xylinum
synthesized a ribbon of cellulose I microfibrils onto a fixed, nematic ordered substrate of glucan chains with unique surface characteristics. The substrate directed the orientation of the motion due to the inverse force of the secretion during biosynthesis, and the microfibrils were aligned along the orientation of the molecular template. Using real-time video analysis, the patterns and rates of deposition were elucidated. Field emission scanning electron microscopy revealed that a strong molecular interaction allowed for the deposition of nascent biosynthesized 3.5-nm cellulose microfibrils with inter-microfibrillar spacings of 7–8 nm on the surface of the template. The cellulose was deposited parallel to the molecular orientation of the template. Directed cellulose synthesis and ordered movement of cells were observed only by using a nematic ordered substrate made from cellulose, and not from ordered crystalline cellulose substrates or ordered cellulose-related synthetic polymers such as polyvinyl alcohol. This unique relationship between directed biosynthesis and the ordered fabrication from the nano to the micro scales could lead to new methodologies for the design of functional materials with desired nanostructures.
PI31 is a modulator of proteasome formation and antigen processing
Authors: Zaiss Dietmar M. W. , Standera Sybille , Kloetzel Peter-M. , Sijts Alice J. A. M.
Published: October 2002

Regulation of the proteasome system, which is responsible for the generation of most MHC class I-bound peptides, occurs through the interaction of the 20S proteasome with several regulatory proteins. One of these is PI31, which actsin vitroas an inhibitor of proteasome activity. Here, we demonstrate that, rather than inhibiting proteasome function, PI31 acts as a selective modulator of the proteasome-mediated steps in MHC class I antigen processing. Overexpression of PI31 in mouse embryonic cells has no impact on proteasome-mediated proteolysis. Instead, PI31, which localizes at the nuclear envelope/endoplasmic reticulum membrane, selectively interferes with the maturation of immunoproteasome precursor complexes. Consequently, overexpression of PI31 abrogates MHC class I presentation of an immunoproteasome-dependent cytotoxic T lymphocyte epitope and reduces the surface MHC class I levels on IFN-γ-treated mouse embryonic cells. Thus, PI31 represents a cellular regulator of proteasome formation and of proteasome-mediated antigen processing.
A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export
Authors: Daelemans Dirk , Afonina Elena , Nilsson Jakob , Werner Gudrun , Kjems Jørgen , De Clercq Erik , Pavlakis George N. , Vandamme Anne-Mieke
Published: October 2002

The HIV-1 Rev protein is an essential regulator of the HIV-1 mRNA expression that promotes the export of unspliced and partially spliced mRNA. The export receptor for the leucine-rich nuclear export signal (NES) of Rev has recently been recognized as CRM1. We identified a low molecular weight compound PKF050-638 as an inhibitor of HIV-1 Rev. This drug inhibits in a dose-dependent fashion Rev-dependent mRNA expression in a cellular assay for Rev function. We show that PKF050-638 is an inhibitor of the CRM1-mediated Rev nuclear export. By using a quantitative
in vitro
CRM1-NES cargo-binding assay, we could demonstrate that PKF050-638 disrupts CRM1-NES interaction. This mode of action is confirmed in cell culture because the drug reversibly interferes with the colocalization of CRM1 and Rev in the nucleolus of the cell. In addition, we prove that the inhibition is through direct interaction of the compound with Cys-539 of CRM1. These effects are similar to those of the known CRM1 inhibitor leptomycin B and suggest that the inhibitory effect of the compound is caused by binding to CRM1 at a similar site. The compound displayed strict structural requirements for its activity, as its enantiomer was inactive in all assays tested. These results show that we identified a drug that interferes with the CRM1-mediated nuclear export of Rev through inhibition of the CRM1-NES complex formation. The reversibility of its binding to CRM1 and its availability through chemical synthesis could make it useful for studying CRM1-mediated export pathways.
Wind-dispersed pollen mediates postglacial gene flow among refugia
Authors: Liepelt Sascha , Bialozyt Ronald , Ziegenhagen Birgit
Published: October 2002

A long-term genetic legacy of refugial isolation has been postulated and was demonstrated for maternal refugial lineages for numerous plant and animal species. The lineages were assumed to have remained separated from each other for several glacial periods. The coniferAbies albaMiller, silver fir, is an excellent model to test whether pollen-mediated gene flow may eliminate the genetic imprints of Pleistocene refugial isolation. Two DNA markers with contrasting modes of inheritance were applied to 100 populations covering the entire range of silver fir in Europe. The markers exhibited each two highly conserved alleles based on an insertion/deletion of 80 bp in the fourth intron of the mitochondrialnad5 gene and on a synonymous substitution in the chloroplastpsbC gene. The geographical distribution of the maternally inherited mitochondrial variation supported the existence of at least two refugia with two recolonizing maternal lineages remaining largely separated throughout the range. The cline of thenad5 allele frequencies was much steeper than the one of the twopsbC alleles. ThepsbC cline was as wide as the whole range of the species. Our results provide striking evidence that even a species with very long generation times and heavy pollen grains was able to establish a highly efficient pollen-mediated gene flow between refugia. Therefore we postulate that an exchange of genetic information between refugia by range-wide paternal introgression is possible in wind-pollinated plant species.
Protection of the intestinal mucosa by intraepithelial γδ T cells
Authors: Chen Yaping , Chou Kevin , Fuchs Elaine , Havran Wendy L. , Boismenu Richard
Published: October 2002

γδ intraepithelial T lymphocytes (IEL) represent a major T cell population within the intestine of unclear functional relevance. The role of intestinal γδ IEL was evaluated in the dextran sodium sulfate (DSS) induced mouse colitis model system. Large numbers of γδ T cells, but not αβ T cells, were localized at sites of DSS-induced epithelial cell damage. γδ IEL in DSS treated mice expressed keratinocyte growth factor (KGF), a potent intestinal epithelial cell mitogen. γδ cell-deficient mice (TCRδ−/−) and KGF-deficient mice (KGF−/−), but not αβ cell-deficient mice (TCRα−/−), were more prone than wild-type mice to DSS-induced mucosal injury and demonstrated delayed tissue repair after termination of DSS treatment. Termination of DSS treatment resulted in vigorous epithelial cell proliferation in wild-type mice but not in TCRδ−/−mice or KGF−/−mice. These results suggest that γδ IEL help preserve the integrity of damaged epithelial surfaces by providing the localized delivery of an epithelial cell growth factor.
Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: The role of NF-κB-repressing factor (NRF) in basal repression of the hiNOS gene
Authors: Feng Xuesheng , Guo Zhong , Nourbakhsh Mahtab , Hauser Hansjorg , Ganster Ray , Shao Lifang , Geller David A.
Published: October 2002

Although nuclear factor (NF)-κB plays a central role in mediating cytokine-stimulated human inducible nitric-oxide synthase (hiNOS) gene transcription, very little is known about the factors involved in silencing of the hiNOS promoter. NF-κB-repressing factor (NRF) interacts with a specific negative regulatory element (NRE) to mediate transcriptional repression of certain NF-κB responsive genes. By sequence comparison with the IFN-β and IL-8 promoters, we identified an NRE in the hiNOS promoter located at −6.7 kb upstream. In A549 and HeLa human cells, constitutive NRF mRNA expression is detected by RT-PCR. Gel shift assay showed constitutive NRF binding to the hiNOS NRE. Mutation of the −6.7-kb NRE site in the hiNOS promoter resulted in loss of NRF binding and increased basal but not cytokine-stimulated hiNOS transcription in promoter transfection experiments. Interestingly, overexpression of NRF suppressed both basal and cytokine-induced hiNOS promoter activity that depended on an intact cis-acting NRE motif. By using stably transformed HeLa cells with the tetracycline on/off expression system, reduction of cellular NRF by expressing antisense NRF increased basal iNOS promoter activity and resulted in constitutive iNOS mRNA expression. These data demonstrate that the transacting NRF protein is involved in constitutive silencing of the hiNOS gene by binding to a cis-acting NRE upstream in the hiNOS promoter.
Selenomethionine regulation of p53 by a ref1-dependent redox mechanism
Authors: Seo Young R. , Kelley Mark R. , Smith Martin L.
Published: October 2002

The cancer chemopreventive properties of selenium compounds are well documented, yet little is known of the mechanism(s) by which these agents inhibit carcinogenesis. We show that selenium in the form of selenomethionine (SeMet) can activate the p53 tumor suppressor protein by a redox mechanism that requires the redox factor Ref1. Assays to measure direct reduction/oxidation of p53 showed a SeMet-dependent response that was blocked by a dominant–negative Ref1. By using a peptide containing only p53 cysteine residues 275 and 277, we demonstrate the importance of these residues in the SeMet-induced response. SeMet induced sequence-specific DNA binding and transactivation by p53. Finally, cellular responses to SeMet were determined in mouse embryo fibroblasts wild-type or null for p53 genes. The evidence suggests that the DNA repair branch of the p53 pathway was activated. The central relevance of DNA repair to cancer prevention is discussed.
Interphase chromosomes in
Arabidopsis
are organized as well defined chromocenters from which euchromatin loops emanate
Authors: Fransz Paul , de Jong J. Hans , Lysak Martin , Castiglione Monica Ruffini , Schubert Ingo
Published: October 2002

Heterochromatin in the model plant
Arabidopsis thaliana
is confined to small pericentromeric regions of all five chromosomes and to the nucleolus organizing regions. This clear differentiation makes it possible to study spatial arrangement and functional properties of individual chromatin domains in interphase nuclei. Here, we present the organization of
Arabidopsis
chromosomes in young parenchyma cells. Heterochromatin segments are organized as condensed chromocenters (CCs), which contain heavily methylated, mostly repetitive DNA sequences. In contrast, euchromatin contains less methylated DNA and emanates from CCs as loops spanning 0.2–2 Mbp. These loops are rich in acetylated histones, whereas CCs contain less acetylated histones. We identified individual CCs and loops by fluorescence
in situ
hybridization by using rDNA clones and 131 bacterial artificial chromosome DNA clones from chromosome 4. CC and loops together form a chromosome territory. Homologous CCs and territories were associated frequently. Moreover, a considerable number of nuclei displayed perfect alignment of homologous subregions, suggesting physical transinteractions between the homologs. The arrangement of interphase chromosomes in
Arabidopsis
provides a well defined system to investigate chromatin organization and its role in epigenetic processes.
Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways
Authors: Twu Cheryl , Liu Nancy Q. , Popik Waldemar , Bukrinsky Michael , Sayre James , Roberts Jaclyn , Rania Shammas , Bramhandam Vishnu , Roos Kenneth P. , MacLellan W. Robb , Fiala Milan
Published: October 2002

We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-α. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-α and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes.HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways.
Growing and navigating the small world Web by local content
Authors: Menczer Filippo
Published: October 2002

Can we model the scale-free distribution of Web hypertext degree under realistic assumptions about the behavior of page authors? Can a Web crawler efficiently locate an unknown relevant page? These questions are receiving much attention due to their potential impact for understanding the structure of the Web and for building better search engines. Here I investigate the connection between the linkage and content topology of Web pages. The relationship between a text-induced distance metric and a link-based neighborhood probability distribution displays a phase transition between a region where linkage is not determined by content and one where linkage decays according to a power law. This relationship is used to propose a Web growth model that is shown to accurately predict the distribution of Web page degree, based on textual content and assuming only local knowledge of degree for existing pages. A qualitatively similar phase transition is found between linkage and semantic distance, with an exponential decay tail. Both relationships suggest that efficient paths can be discovered by decentralized Web navigation algorithms based on textual and/or categorical cues.
Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton
Authors: Shupliakov Oleg , Bloom Ona , Gustafsson Jenny S. , Kjaerulff Ole , Löw Peter , Tomilin Nikolay , Pieribone Vincent A. , Greengard Paul , Brodin Lennart
Published: October 2002

Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of
Clostridium botulinum
C2 toxin, and
N
-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of
N
-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster.
Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases
Authors: Schwarzer Dirk , Mootz Henning D. , Linne Uwe , Marahiel Mohamed A.
Published: October 2002

Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEII
srf
) and bacitracin (TEII
bac
). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4′-phosphopantetheine (4′PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEII
srf
, a
K
M
of 0.9 μM and a
k
cat
of 95 min
−1
was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEII
srf
. These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4′PP transferases that use not only free CoA, but also acyl-CoAs as 4′PP donors.
Hematopoietic-specific activators establish an overlapping pattern of histone acetylation and methylation within a mammalian chromatin domain
Authors: Kiekhaefer Carol M. , Grass Jeffrey A. , Johnson Kirby D. , Boyer Meghan E. , Bresnick Emery H.
Published: October 2002

Posttranslational modification of histones through acetylation, methylation, and phosphorylation is a common mode of regulating chromatin structure and, therefore, diverse nuclear processes. One such modification, methylated histone H3 at lysine-4 (H3-meK4), colocalizes with hyperacetylated histones H3 and H4 in mammalian chromatin. Whereas activators directly recruit acetyltransferases, the process whereby H3-meK4 is established is unknown. We tested whether the hematopoietic-specific activators NF-E2 and GATA-1, which mediate transactivation of the β-globin genes, induce both histone acetylation and H3-meK4. Through the use of NF-E2- and GATA-1-null cell lines, we show that both activators induce H3 acetylation at the promoter upon transcriptional activation. However, analysis of H3-mek4 revealed that NF-E2 and GATA-1 differentially regulate chromatin modifications at the β
major
promoter. NF-E2, but not GATA-1, induces H3-meK4 at the promoter. Thus, under conditions in which NF-E2 and GATA-1 activate the transcription of an endogenous gene at least 570-fold, these activators differ in their capacity to induce H3-meK4. Despite strong H3-meK4 at hypersensitive site 2 of the upstream locus control region, neither factor was required to establish H3-meK4 at this site. These results support a model in which multiple tissue-specific activators collectively function to assemble a composite histone modification pattern, consisting of overlapping histone acetylation and methylation. As GATA-1 induced H3 acetylation, but not H3-meK4, at the promoter, H3 acetylation and H3-meK4 components of a composite histone modification pattern can be established independently.
Molecular topography imaging by intermembrane fluorescence resonance energy transfer
Authors: Wong Amy P. , Groves Jay T.
Published: October 2002

Fluorescence resonance energy transfer (FRET) between lipid-linked donor and acceptor molecules in two apposing lipid bilayer membranes is used to resolve topographical features at an intermembrane junction. Efficient energy transfer occurs when the membranes are apposed closely, which creates an image, or footprint, that maps the contact zone and reveals nanometer-scale topographical structures. We experimentally characterize intermembrane FRET by using a supported membrane junction consisting of a glass-supported lipid membrane, onto which a second membrane is deposited by rupture of a giant vesicle. A series of membrane junctions containing different glycolipids (phosphatidylinositol and ganglioside G
M1
), protein (cholera toxin), and lipid-linked polyethylene glycol are studied. The carbohydrate and protein components influence the intermembrane separation. Differential FRET efficiency is clearly distinguishable for each case. Quantitative analysis of the FRET efficiency yields measurements of intermembrane-separation distances that agree precisely with structural data on G
M1
and cholera toxin. The lateral arrangement of molecular species on the membrane surface thus can be discerned by their influence on membrane spacing without the need for direct labeling of the molecule of interest. In the case of polyethylene glycol lipid-containing membrane junctions, imaging by intermembrane FRET reveals spontaneously forming patterns that are not visible in conventional fluorescence images.
Solving the freeloaders paradox: Genetic associations and frequency-dependent selection in the evolution of cooperation among nonrelatives
Authors: Avilés Leticia
Published: October 2002

One of the enduring problems in the study of social evolution has been to understand how cooperation can be maintained in the presence of freeloaders, individuals that take advantage of the more cooperative members of groups they are eager to join. The freeloader problem has been particularly troublesome when groups consist of nonrelatives, and no inclusive fitness benefits accrue to individuals that contribute more heavily to communal activities. These theoretical difficulties, however, are not mirrored by the numerous examples of cooperative or even altruistic behaviors exhibited by groups of nonrelatives in nature (e.g., many human groups, communally nesting bees, multiple queen-founding ants, cellular slime molds, and social bacteria). Using a model in which cooperation and grouping tendencies are modeled as coevolving dynamical variables, I show that the freeloader problem can be addressed when group-size effects on fitness are considered explicitly. I show that freeloaders, whose presence is reflected in the development of linkage disequilibrium between grouping and cooperation, increase in frequency when rare, but are selected against when common due to the reduced productivity of the groups they overburden with their presence. Freeloader frequencies thus periodically rise and fall around an equilibrium shown here to be dynamic. These results highlight the importance of group-level effects in the origin and maintenance of sociality, illustrate the dynamic nature of equilibria when multiple levels of selection are involved, and provide a solution to the freeloaders paradox.
DysregulatedTCL1promotes multiple classes of mature B cell lymphoma
Authors: Hoyer Katrina K. , French Samuel W. , Turner Devin E. , Nguyen Mai T. N. , Renard Mathilde , Malone Cindy S. , Knoetig Sonja , Qi Chen-Feng , Su Thomas T. , Cheroutre Hilde , Wall Randolph , Rawlings David J. , Morse Herbert C. , Teitell Michael A.
Published: October 2002

TheTCL1protooncogene is overexpressed in many mature B cell lymphomas, especially from AIDS patients. To determine whether aberrant expression promotes B cell transformation, we generated a murine model in which aTCL1transgene was overexpressed at similar levels in both B and T cells. Strikingly, transgenic mice developed Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma (DLBCL) with attendant Bcl-6 expression and mutated JHgene segments at a very high penetrance beginning at 4 months of age. In contrast, only one mouse developed a T cell malignancy at 15 months, consistent with a longer latency for transformation of T cells byTCL1. Activation of premalignant splenic B cells by means of B cell antigen receptor (BCR) engagement resulted in significantly increased proliferation and augmented AKT-dependent signaling, including increased S6 ribosomal protein phosphorylation. Transgenic spleen cells also survived longer than wild-type spleen cells in long-term culture. Together these data demonstrate thatTCL1is a powerful oncogene that, when overexpressed in both B and T cells, predominantly yields mature B cell lymphomas.
Heterochromatin protein 2 (HP2), a partner of HP1 in
Drosophila
heterochromatin
Authors: Shaffer Christopher D. , Stephens Gena E. , Thompson Brandi A. , Funches Levi , Bernat John A. , Craig Carolyn A. , Elgin Sarah C. R.
Published: October 2002

Heterochromatin protein 1 (HP1), first discovered in
Drosophila melanogaster
, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing.
Three-dimensional model of the human platelet integrin α
IIb
β
3
based on electron cryomicroscopy and x-ray crystallography
Authors: Adair Brian D. , Yeager Mark
Published: October 2002

Integrins are a large family of heterodimeric transmembrane signaling proteins that affect diverse biological processes such as development, angiogenesis, wound healing, neoplastic transformation, and thrombosis. We report here the three-dimensional structure at 20-Å resolution of the unliganded, low-affinity state of the human platelet integrin α
IIb
β
3
derived by electron cryomicroscopy and single particle image reconstruction. The large ectodomain and small cytoplasmic domains are connected by a rod of density that we interpret as two parallel transmembrane α-helices. The docking of the x-ray structure of the α
V
β
3
ectodomain into the electron cryomicroscopy map of α
IIb
β
3
requires hinge movements at linker regions between domains in the crystal structure. Comparison of the putative high- and low-affinity conformations reveals dramatic conformational changes associated with integrin activation.
Permanent and panerythroid correction of murine β thalassemia by multiple lentiviral integration in hematopoietic stem cells
Authors: Imren Suzan , Payen Emmanuel , Westerman Karen A. , Pawliuk Robert , Fabry Mary E. , Eaves Connie J. , Cavilla Benjamin , Wadsworth Louis D. , Beuzard Yves , Bouhassira Eric E. , Russell Robert , London Irving M. , Nagel Ronald L. , Leboulch Philippe , Humphries R. Keith
Published: October 2002

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid β globin gene expression has not yet been achieved in β thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe β thalassemia in mice, resulting from a homozygous deletion of the β major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [β globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time ≈95% of the red blood cells in all mice contained human β globin contributing to 32 ± 4% of all β-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free α globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in β thalassemia patients.
Effect of polyethylene glycol on the liquid–liquid phase transition in aqueous protein solutions
Authors: Annunziata Onofrio , Asherie Neer , Lomakin Aleksey , Pande Jayanti , Ogun Olutayo , Benedek George B.
Published: October 2002

We have studied the effect of polyethylene glycol (PEG) on the liquid–liquid phase separation (LLPS) of aqueous solutions of bovine γD-crystallin (γD), a protein in the eye lens. We observe that the phase separation temperature increases with both PEG concentration and PEG molecular weight. PEG partitioning, which is the difference between the PEG concentration in the two coexisting phases, has been measured experimentally and observed to increase with PEG molecular weight. The measurements of both LLPS temperature and PEG partitioning in the ternary γD-PEG-water systems are used to successfully predict the location of the liquid–liquid phase boundary of the binary γD-water system. We show that our LLPS measurements can be also used to estimate the protein solubility as a function of the concentration of crystallizing agents. Moreover, the slope of the tie-lines and the dependence of LLPS temperature on polymer concentration provide a powerful and sensitive check of the validity of excluded volume models. Finally, we show that the increase of the LLPS temperature with PEG concentration is due to attractive protein–protein interactions.
A GDP/GTP exchange protein for the Rab3 small G protein family up-regulates a postdocking step of synaptic exocytosis in central synapses
Authors: Yamaguchi Kazuhiko , Tanaka Miki , Mizoguchi Akira , Hirata Yoshihiro , Ishizaki Hiroyoshi , Kaneko Kumi , Miyoshi Jun , Takai Yoshimi
Published: October 2002

The Rab3 GDP/GTP exchange protein (Rab3 GEP) activates the Rab3 small GTP-binding protein (G protein) family, including Rab3A that is an important member controlling synaptic vesicle trafficking. Here, we examined the role of Rab3 GEP in regulating neurotransmitter release in autapses of mouse hippocampal neurons in culture. The release probability was markedly reduced in Rab3 GEP−/− neurons, whereas the readily releasable pool size was not different between WT and Rab3 GEP−/− neurons, indicating that Rab3 GEP up-regulates a postdocking step of synaptic exocytosis. Because Rab3A reportedly down-regulates Ca
2+
-triggered fusion of synaptic vesicles, these results provide evidence for a role of Rab3 GEP in the postdocking process distinct from Rab3A activation.
Common features in structures and sequences of sandwich-like proteins
Authors: Kister Alexander E. , Finkelstein Alexei V. , Gelfand Israel M.
Published: October 2002

The goal of this work is to define the structural and sequence features common to sandwich-like proteins (SPs), a group of very different proteins now comprising 69 superfamilies in 38 protein folds. Analysis of the arrangements of strands within main sandwich sheets revealed a rigorously defined constraint on the supersecondary substructure that holds true for 94% of known SP structures. The invariant substructure consists of two interlocked pairs of neighboring β-strands. It is even more typical for centers of SP than the well-known “Greek key” strands arrangement for their edges. As homology among these proteins is not usually detectable even with the most powerful sequence-comparing algorithms, we employed a structure-based approach to sequence alignment. Within the interlocked strands we found 12 positions with fixed structural roles in SP. A residue at any of these positions possesses similar structural properties with residues in the same position of other SPs. The 12 positions lie at the center of the interface between the β-sheets and form the common geometrical core of SPs. Of the 12 positions, 8 are occupied by only four hydrophobic residues in 80% of all SPs.
Promiscuity in ligand-binding: The three-dimensional structure of a
Piromyces
carbohydrate-binding module, CBM29-2, in complex with cello- and mannohexaose
Authors: Charnock Simon J. , Bolam David N. , Nurizzo Didier , Szabó Lóránd , McKie Vincent A. , Gilbert Harry J. , Davies Gideon J.
Published: October 2002

Carbohydrate–protein recognition is central to many biological processes. Enzymes that act on polysaccharide substrates frequently contain noncatalytic domains, “carbohydrate-binding modules” (CBMs), that target the enzyme to the appropriate substrate. CBMs that recognize specific plant structural polysaccharides are often able to accommodate both the variable backbone and the side-chain decorations of heterogeneous ligands. “CBM29” modules, derived from a noncatalytic component of the
Piromyces equi
cellulase/hemicellulase complex, provide an example of this selective yet flexible recognition. They discriminate strongly against some polysaccharides while remaining relatively promiscuous toward both β-1,4-linked manno- and cello-oligosaccharides. This feature may reflect preferential, but flexible, targeting toward glucomannans in the plant cell wall. The three-dimensional structure of CBM29-2 and its complexes with cello- and mannohexaose reveal a β-jelly-roll topology, with an extended binding groove on the concave surface. The orientation of the aromatic residues complements the conformation of the target sugar polymer while accommodation of both manno- and gluco-configured oligo- and polysaccharides is conferred by virtue of the plasticity of the direct interactions from their axial and equatorial 2-hydroxyls, respectively. Such flexible ligand recognition targets the anaerobic fungal complex to a range of different components in the plant cell wall and thus plays a pivotal role in the highly efficient degradation of this composite structure by the microbial eukaryote.