bluuebunny/crossref_metadata_2025 huggingface dataset:
Strong selective sweep associated with a transposon insertion inDrosophila simulans
Authors: Schlenke Todd A. , Begun David J.
Published: February 2004

We know little about several important properties of beneficial mutations, including their mutational origin, their phenotypic effects (e.g., protein structure changes vs. regulatory changes), and the frequency and rapidity with which they become fixed in a population. One signature of the spread of beneficial mutations is the reduction of heterozygosity at linked sites. Here, we present population genetic data from several loci across chromosome arm 2R inDrosophila simulans. A 100-kb segment from a freely recombining region of this chromosome shows extremely reduced heterozygosity in a California population sample, yet typical levels of divergence between species, suggesting that at least one episode of strong directional selection has occurred in the region. The 5′ flanking sequence of one gene in this region,Cyp6g1(a cytochrome P450), is nearly fixed for aDoctransposable element insertion. Presence of the insertion is correlated with increased transcript abundance ofCyp6g1, a phenotype previously shown to be associated with insecticide resistance inDrosophila melanogaster. Surveys of nucleotide variation in the same genomic region in an AfricanD. simulanspopulation revealed no evidence for a high-frequencyDocelement and no evidence for reduced polymorphism. These data are consistent with the notion that theDocelement is a geographically restricted beneficial mutation. Data fromD. simulans Cyp6g1are paralleled in many respects by data from its sister speciesD. melanogaster.
Evolution of the
MAT
locus and its Ho endonuclease in yeast species
Authors: Butler Geraldine , Kenny Claire , Fagan Ailís , Kurischko Cornelia , Gaillardin Claude , Wolfe Kenneth H.
Published: February 2004

The genetics of the mating-type (
MAT
) locus have been studied extensively in
Saccharomyces cerevisiae
, but relatively little is known about how this complex system evolved. We compared the organization of
MAT
and mating-type-like (
MTL
) loci in nine species spanning the hemiascomycete phylogenetic tree. We inferred that the system evolved in a two-step process in which silent
HMR
/
HML
cassettes appeared, followed by acquisition of the Ho endonuclease from a mobile genetic element. Ho-mediated switching between an active
MAT
locus and silent cassettes exists only in the
Saccharomyces sensu stricto
group and their closest relatives:
Candida glabrata, Kluyveromyces delphensis
, and
Saccharomyces castellii
. We identified
C. glabrata MTL1
as the ortholog of the
MAT
locus of
K. delphensis
and show that switching between
C. glabrata MTL1
a and
MTL1
α genotypes occurs
in vivo
. The more distantly related species
Kluyveromyces lactis
has silent cassettes but switches mating type without the aid of Ho endonuclease. Very distantly related species such as
Candida albicans
and
Yarrowia lipolytica
do not have silent cassettes. In
Pichia angusta
, a homothallic species, we found
MAT
α
2, MAT
α
1
, and
MAT
a1 genes adjacent to each other on the same chromosome. Although some continuity in the chromosomal location of the
MAT
locus can be traced throughout hemiascomycete evolution and even to
Neurospora
, the gene content of the locus has changed with the loss of an HMG domain gene (
MAT
a2) from the
MAT
a idiomorph shortly after
HO
was recruited.
Imaging reversal of multidrug resistance in living mice with bioluminescence:
MDR1
P-glycoprotein transports coelenterazine
Authors: Pichler Andrea , Prior Julie L. , Piwnica-Worms David
Published: February 2004

Coelenterazine is widely distributed among marine organisms, producing bioluminescence by calcium-insensitive oxidation mediated by
Renilla
luciferase (Rluc) and calcium-dependent oxidation mediated by the photoprotein aequorin. Despite its abundance in nature and wide use of both proteins as reporters of gene expression and signal transduction, little is known about mechanisms of coelenterazine transport and cell permeation. Interestingly, coelenterazine analogues share structural and physiochemical properties of compounds transported by the multidrug resistance
MDR1
P-glycoprotein (Pgp). Herein, we report that living cells stably transfected with a codon-humanized
Rluc
show coelenterazine-mediated bioluminescence in a highly
MDR1
Pgp-modulated manner. In Pgp-expressing
Rluc
cells, low baseline bioluminescence could be fully enhanced (reversed) to non-Pgp matched control levels with potent and selective Pgp inhibitors. Therefore, using coelenterazine and noninvasive bioluminescence imaging
in vivo
, we could directly monitor tumor-specific Pgp transport inhibition in living mice. While enabling molecular imaging and high-throughput screening of drug resistance pathways, these data also raise concern for the indiscriminate use of
Rluc
and
aequorin
as reporters in intact cells or transgenic animals, wherein Pgp-mediated alterations in coelenterazine permeability may impact results.
An
Arabidopsis
indole-3-butyric acid-response mutant defective in PEROXIN6, an apparent ATPase implicated in peroxisomal function
Authors: Zolman Bethany K. , Bartel Bonnie
Published: February 2004

Genetic evidence suggests that plant peroxisomes are the site of fatty acid β-oxidation and conversion of the endogenous auxin indole-3-butyric acid (IBA) to the active hormone indole-3-acetic acid.
Arabidopsis
mutants that are IBA resistant and sucrose dependent during early development are likely to have defects in β-oxidation of both IBA and fatty acids. Several of these mutants have lesions in peroxisomal protein genes. Here, we describe the
Arabidopsis pex6
mutant, which is resistant to the inhibitory effects of IBA on root elongation and the stimulatory effects of IBA on lateral root formation.
pex6
also is sucrose dependent during early seedling development and smaller and more pale green than WT throughout development.
PEX6
encodes an apparent ATPase similar to yeast and human proteins required for peroxisomal biogenesis, and a human
PEX6
cDNA can rescue the
Arabidopsis pex6
mutant. The
pex6
mutant has reduced levels of the peroxisomal matrix protein receptor PEX5, and
pex6
defects can be partially rescued by
PEX5
overexpression. These results suggest that PEX6 may facilitate PEX5 recycling and thereby promote peroxisomal matrix protein import.
Saccharomyces cerevisiae
Sin3p facilitates DNA double-strand break repair
Authors: Jazayeri Ali , McAinsh Andrew D. , Jackson Stephen P.
Published: February 2004

There are two main pathways in eukaryotic cells for the repair of DNA double-strand breaks: homologous recombination and nonhomologous end joining. Because eukaryotic genomes are packaged in chromatin, these pathways are likely to require the modulation of chromatin structure. One way to achieve this is by the acetylation of lysine residues on the N-terminal tails of histones. Here we demonstrate that Sin3p and Rpd3p, components of one of the predominant histone deacetylase complexes of
Saccharomyces cerevisiae
, are required for efficient nonhomologous end joining. We also show that lysine 16 of histone H4 becomes deacetylated in the proximity of a chromosomal DNA double-strand break in a Sin3p-dependent manner. Taken together, these results define a role for the Sin3p/Rpd3p complex in the modulation of DNA repair.
Histone H3 lysine 9 methylation is required for DNA elimination in developing macronuclei in
Tetrahymena
Authors: Liu Yifan , Mochizuki Kazufumi , Gorovsky Martin A.
Published: February 2004

Genome-wide DNA elimination accompanies development of the somatic macronucleus from the germ-line micronucleus during the sexual process of conjugation in the ciliated protozoan
Tetrahymena thermophila
. Small RNAs, referred to as “scan RNAs” (scnRNAs), that accumulate only during conjugation are highly enriched in the eliminated sequences, and mutations that prevent DNA elimination also affect the accumulation of scnRNAs, suggesting that an RNA interference (RNAi)-like mechanism is involved in DNA elimination. Histone H3 that is methylated at lysine 9 (K9) is a hallmark of heterochromatin and, in
Tetrahymena
, is found only in developing macronuclei (anlagen) in association with chromatin containing the sequences undergoing elimination. In this article, we demonstrate that a mutation in the
TWI1
gene that eliminates the accumulation of scnRNAs also abolishes H3 methylation at K9. We created mutant strains of
Tetrahymena
in which the only major H3 contained a K9Q mutation. These mutants accumulated scnRNAs normally during conjugation but showed dramatically reduced efficiency of DNA elimination. These results provide strong genetic evidence linking an RNAi-like pathway, H3 K9 methylation, and DNA elimination in
Tetrahymena
.
The endogenous retroviral locus ERVWE1 is a bona fide gene involved in hominoid placental physiology
Authors: Mallet François , Bouton Olivier , Prudhomme Sarah , Cheynet Valérie , Oriol Guy , Bonnaud Bertrand , Lucotte Gérard , Duret Laurent , Mandrand Bernard
Published: February 2004

The definitive demonstration of a role for a recently acquired gene is a difficult task, requiring exhaustive genetic investigations and functional analysis. The situation is indeed much more complicated when facing multicopy gene families, because most or portions of the gene are conserved among the hundred copies of the family. This is the case for the ERVWE1 locus of the human endogenous retrovirus W family (HERV-W), which encodes an envelope glycoprotein (syncytin) likely involved in trophoblast differentiation. Here we describe, in 155 individuals, the positional conservation of this locus and the preservation of the envelope ORF. Sequencing of the critical elements of the ERVWE1 provirus showed a striking conservation among the 48 alleles of 24 individuals, including the LTR elements involved in the transcriptional machinery, the splice sites involved in the maturation of subgenomic Env mRNA, and the Env ORF. The functionality and tissue specificity of the 5′ LTR were demonstrated, as well as the fusogenic activity of the envelope polymorphic variants. Such functions were also shown to be preserved in the orthologous loci isolated from chimpanzee, gorilla, orangutan, and gibbon. This functional preservation among humans and during evolution strongly argued for the involvement of this recently acquired retroviral envelope glycoprotein in hominoid placental physiology.
The metabolic world of
Escherichia coli
is not small
Authors: Arita Masanori
Published: February 2004

To elucidate the organizational and evolutionary principles of the metabolism of living organisms, recent studies have addressed the graph-theoretic analysis of large biochemical networks responsible for the synthesis and degradation of cellular building blocks [Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabási, A. L. (2000)
Nature
407, 651-654; Wagner, A. & Fell, D. A. (2001)
Proc. R. Soc. London Ser. B
268, 1803-1810; and Ma, H.-W. & Zeng, A.-P. (2003)
Bioinformatics
19, 270-277]. In such studies, the global properties of the network are computed by considering enzymatic reactions as links between metabolites. However, the pathways computed in this manner do not conserve their structural moieties and therefore do not correspond to biochemical pathways on the traditional metabolic map. In this work, we reassessed earlier results by digitizing carbon atomic traces in metabolic reactions annotated for
Escherichia coli
. Our analysis revealed that the average path length of its metabolism is much longer than previously thought and that the metabolic world of this organism is not small in terms of biosynthesis and degradation.
In vitro
synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state
Authors: Elbaz Yael , Steiner-Mordoch Sonia , Danieli Tsafi , Schuldiner Shimon
Published: February 2004

EmrE is a small multidrug transporter from
Escherichia coli
that provides a unique model for the study of polytopic membrane proteins. Here, we show its synthesis in a cell-free system in a fully functional form. The detergent-solubilized protein binds substrates with high affinity and, when reconstituted into proteoliposomes, transports substrate in a Δμ̃
H
+
-dependent fashion. Here, we used the cell-free system to study the oligomeric properties of EmrE. EmrE functions as an oligomer, but the size of the functional oligomer has not been established unequivocally. Coexpression of two plasmids in the cell-free system allowed demonstration of functional complementation and pull-down experiments confirmed that the basic functional unit is the dimer. An additional interaction between dimers has been detected by using crosslinking between unique Cys residues. This finding implies the existence of a dimer of dimers.
Increased activity of antagonists of growth hormone-releasing hormone substituted at positions 8, 9, and 10
Authors: Varga Jozsef L. , Schally Andrew V. , Horvath Judit E. , Kovacs Magdolna , Halmos Gabor , Groot Kate , Toller Gabor L. , Rekasi Zoltan , Zarandi Marta
Published: February 2004

Antagonists of human growth hormone-releasing hormone (hGHRH) with increased potency and improved enzymatic and chemical stability are needed for potential clinical applications. We synthesized 21 antagonistic analogs of hGHRH(1-29)NH2, substituted at positions 8, 9, and 10 of the common core sequence {phenylacetyl-Tyr1,d-Arg2,28,para-chloro-phenylalanine 6, Arg9/homoarginine 9, Tyr10/O-methyltyrosine 10, α-aminobutyric acid 15, norleucine 27, Har29} hGHRH(1-29)NH2. Inhibitory effects on hGHRH-induced GH release were evaluatedin vitroin a superfused rat pituitary system, as well asin vivoafter i.v. injection into rats. The binding affinities of the peptides to pituitary GHRH receptors were also determined. Introduction ofpara-amidinophenylalanine 10 yielded antagonists JV-1-62 and -63 with the highest activitiesin vitroand lowest receptor dissociation constants (Ki= 0.057-0.062 nM). Antagonists JV-1-62 and -63 also exhibited the strongest effectin vivo, significantly (P< 0.05-0.001) inhibiting hGHRH-induced GH release for at least 1 h.Para-aminophenylalanine 10 andO-ethyltyrosine 10 substitutions yielded antagonists potentin vitro, but His10, 3,3′-diphenylalanine 10, 2-naphthylalanine 10, and cyclohexylalanine 10 modifications were detrimental. Antagonists containing citrulline 9 (in MZ-J-7-72), amidinophenylalanine 9 (in JV-1-65), His9,d-Arg9, citrulline 8, Ala8,d-Ala8, or α-aminobutyric acid 8 substituents also had high activity and receptor affinityin vitro. However,in vitropotencies of analogs with substitution in position 9 correlated poorly with acute endocrine effectsin vivo, as exemplified by the weak and/or short inhibitory actions of antagonists JV-1-65 and MZ-J-7-72 on GH releasein vivo. Nevertheless, antagonist JV-1-65 was more potent than JV-1-63 in tests on inhibition of the growth of human prostatic and lung cancer lines xenografted into nude mice. This indicates that oncological activity may be based on several mechanisms. hGHRH antagonists with improved efficacy could be useful for treatment of cancers that depend on insulin-like growth factors or GHRH.
Protein phosphorylation can regulate metabolite concentrations rather than control flux: The example of glycogen synthase
Authors: Schafer James R. A. , Fell David A. , Rothman Douglas , Shulman Robert G.
Published: February 2004

Despite dramatic increases in glucose influx during the transition from fasting to fed states, plasma glucose concentration remains tightly controlled. This constancy is in large part due to the capacity of skeletal muscle to absorb excess glucose and store it as glycogen. The magnitude of this capacity is controlled by insulin by way of regulated insertion of glucose transporters into the muscle cell membrane. Here, we examine the mechanism by which muscle cells are able to tolerate large flux increases across their transporters without significantly changing their own metabolite pools. MCA was used to probe data sets that measured the effects of changing plasma glucose and/or insulin concentrations on the rates of glycogen synthesis and the concentrations of metabolites, particularly glucose-6-phosphate. We find that homeostasis is achieved by insulin-dependent phosphorylation changes in GSase sensitivity to the upstream metabolite glucose-6-phosphate. The centrality of GSase to homeostasis resolves the paradox of its sensitivity to allosteric and covalent regulation despite its minimal role in flux control. The importance of this role for enzymatic phosphorylation to diabetes pathology is discussed, and its general applicability is suggested.
Crystal and molecular structure of a benzo[
a
]pyrene 7,8-diol 9,10-epoxide
N
2
-deoxyguanosine adduct: Absolute configuration and conformation
Authors: Karle Isabella L. , Yagi Haruhiko , Sayer Jane M. , Jerina Donald M.
Published: February 2004

Benzo[
a
]pyrene 7,8-diol 9,10-epoxide adducts in DNA are implicated in mutagenesis, and their formation from the diol epoxides and subsequent incorrect replication by human DNA polymerases provide an attractive mechanism for the induction of cancer by this highly carcinogenic hydrocarbon and its diol epoxide metabolites. Here, we describe the crystal structure of such an adduct at the exocyclic amino group of a purine nucleoside. The present adduct derives from trans opening at C10 of the (-)-(7
S
,8
R
)-diol (9
R
,10
S
)-epoxide enantiomer by the exocyclic
N
2
-amino group of deoxyguanosine. In the crystal, the pyrene rings of adjacent molecules stack with each other, but the guanine bases do not stack either intermolecularly with each other or intramolecularly with the pyrene. The most notable features of the molecular structure are (
i
) independent and unambiguous proof of the absolute configuration of the adduct based on the spatial relationship between the known chiral carbon atoms of the deoxyribose and the four asymmetric centers in the hydrocarbon moiety; (
ii
) visualization of the relative orientations of the pyrene and guanine ring systems as well as the conformation of the partially saturated hydrocarbon ring (comprising carbon atoms 7, 8, 9, and 10), both of which conformational features in the crystal are in good agreement with deductions from NMR and CD measurements in solution; and (
iii
) the presence in the crystal of a
syn
glycosidic torsion angle, a conformation that is unusual in B-DNA but that may be involved in error-prone replication of these benzo[
a
]pyrene 7,8-diol 9,10-epoxide deoxyguanosine adducts by DNA polymerases.
Prehistoric Inuit whalers affected Arctic freshwater ecosystems
Authors: Douglas Marianne S. V. , Smol John P. , Savelle James M. , Blais Jules M.
Published: February 2004

It is commonly assumed that High Arctic lakes and ponds were not affected by direct local human activities before the arrival of Europeans, because most native peoples were primarily nomadic, maintained relatively low population densities, and practiced unintrusive hunting and gathering technologies. Our archeological and paleolimnological data show that this was not always the case. Thule Inuit whalers, whose winter settlements consisted of houses constructed from the bones of bowhead whales on Somerset Island between about
anno Domini
1200 and 1600, markedly changed pond water quality and ecology. The arrival of whalers 8 centuries ago caused marked changes in water chemistry and the expansion of moss substrates. Although whalers abandoned the area >4 centuries ago, the legacy of these human disturbances is still evident in the pond's present-day limnology and is characterized by elevated nutrient concentrations and atypical biota. This is the earliest reported paleolimnological record of changes in aquatic ecology associated with local human activities in Canada or the United States, or for any circumpolar ecosystem.
Electrical wound-healing assay for cells
in vitro
Authors: Keese Charles R. , Wegener Joachim , Walker Sarah R. , Giaever Ivar
Published: February 2004

Confluent cell monolayers in tissue culture are fragile and can easily be mechanically disrupted, often leaving an area devoid of cells. This opening in the cell sheet is then repopulated, because the cells on the fringe of the damage, which are no longer contact-inhibited, move into the available space. This mechanical disruption is often done deliberately in a “wound-healing” assay as a means to assess the migration of the cells. In such assays, a scrape is made in the cell layer followed by microscopy to monitor the advance of the cells into the wound. We have found that these types of assays can also be accomplished electrically. In this approach, cells growing on small electrodes and monitored by using electric cell-substrate impedance sensing are subjected to currents, resulting in severe electroporation and subsequent cell death. After this invasive treatment, the electrode's impedance is again monitored to chart the migration and ultimate healing of the wound. We report here that this procedure to study cell behavior is both highly reproducible, quantitative, and provides data similar to that acquired with traditional measurements.
ELG1
, a regulator of genome stability, has a role in telomere length regulation and in silencing
Authors: Smolikov Sarit , Mazor Yuval , Krauskopf Anat
Published: February 2004

Telomeres, the natural ends of eukaryotic chromosomes, prevent the loss of chromosomal sequences and preclude their recognition as broken DNA. Telomere length is kept under strict boundaries by the action of various proteins, some with negative and others with positive effects on telomere length. Recently, data have been accumulating to support a role for DNA replication in the control of telomere length, although through a currently poorly understood mechanism. Elg1p, a replication factor C (RFC)-like protein of yeast, contributes to genome stability through a putative replication-associated function. Here, we show that Elg1p participates in negative control of telomere length and in telomeric silencing through a replication-mediated pathway. We show that the telomeric function of Elg1 is independent of recombination and completely dependent on an active telomerase. Additionally, this function depends on yKu and DNA polymerase. We discuss alternative models to explain how Elg1p affects telomere length.
Production of fertile offspring from genetically infertile male mice
Authors: Yanagimachi Ryuzo , Wakayama Teruhiko , Kishikawa Hidefumi , Fimia Gian Maria , Monaco Lucia , Sassone-Corsi Paolo
Published: February 2004

A number of recessive autosomal genes cause male infertility. Male mice homozygous for the blind-sterile (bs/bs) and quaking-sterile (qk/qk) gene mutations are sterile, because they either do not produce any spermatozoa or produce only a few abnormal spermatozoa. Mice lacking the cyclic AMP responsive-element modulator gene are sterile due to failure of spermiogenesis. All these mice, however, are able to produce fertile offspring when their spermatozoa or round spermatids are injected into oocytes of normal females. This implies that genetic and epigenetic elements necessary for syngamy and embryonic development are established in round spermatids and spermatozoa of these animals, even though their spermatogenic cells are destined to die (bs/bsandqk/qk) or are programmed to undergo apoptosis (cyclic AMP responsive-element modulator-null) without becoming functional spermatozoa.
Airborne signals prime plants against insect herbivore attack
Authors: Engelberth Juergen , Alborn Hans T. , Schmelz Eric A. , Tumlinson James H.
Published: February 2004

Green leafy volatiles (GLV), six-carbon aldehydes, alcohols, and esters commonly emitted by plants in response to mechanical damage or herbivory, induced intact undamaged corn seedlings to rapidly produce jasmonic acid (JA) and emit sesquiterpenes. More importantly, corn seedlings previously exposed to GLV from neighboring plants produced significantly more JA and volatile sesquiterpenes when mechanically damaged and induced with caterpillar regurgitant than seedlings not exposed to GLV. The use of pure synthetic chemicals revealed that (
Z
)
-
3-hexenal, (
Z
)
-
3-hexen-1-ol, and (
Z
)
-
3-hexenyl acetate have nearly identical priming activity. Caterpillar-induced nocturnal volatiles, which are enriched in GLV, also exhibited a strong priming effect, inducing production of larger amounts of JA and release of greater quantities of volatile organic compounds after caterpillar regurgitant application. In contrast, GLV priming did not affect JA production induced by mechanical wounding alone. Thus, GLV specifically prime neighboring plants against impending herbivory by enhancing inducible chemical defense responses triggered during attack and may play a key role in plant-plant signaling and plant-insect interactions.
Stabilities of folding of clustered, two-repeat fragments of spectrin reveal a potential hinge in the human erythroid spectrin tetramer
Authors: MacDonald Ruby I. , Cummings Julie A.
Published: February 2004

The large size of spectrin, the flexible protein promoting reversible deformation of red cells, has been an obstacle to elucidating the molecular mechanism of its function. By studying cloned fragments of the repeating unit domain, we have found a correspondence between positions of selected spectrin repeats in a tetramer with their stabilities of folding. Six fragments consisting of two spectrin repeats were selected for study primarily on the basis of the predicted secondary structures of their linker regions. Fragments with a putatively helical linker were more stable to urea- and heat-induced unfolding than those with a putatively nonhelical linker. Two of the less stably folded fragments, human erythroid α-spectrin repeats 13 and 14 (HEα13,14) and human erythroid β-spectrin repeats 8 and 9 (HEβ8,9), are located opposite each other on antiparallel spectrin dimers. At least partial unfolding of these repeats under physiological conditions indicates that they may serve as a hinge. Also less stably folded, the fragment of human erythroid α-spectrin repeats 4 and 5 (HEα4,5) lies opposite the site of interaction between the partial repeats at the C- and N-terminal ends of β- and α-spectrin, respectively, on the opposing dimer. More stably folded fragments, human erythroid α-spectrin repeats 1 and 2 (HEα1,2) and human erythroid α-spectrin repeats 2 and 3 (HEα2,3), lie nearly opposite each other on antiparallel spectrin dimers of a tetramer. These clusterings along the spectrin tetramer of repeats with similar stabilities of folding may have relevance for spectrin function, particularly for its well known flexibility.
The 1.8-Å resolution crystal structure of YDR533Cp fromSaccharomyces cerevisiae: A member of the DJ-1/ThiJ/PfpI superfamily
Authors: Wilson Mark A. , Amour Courtney V. St. , Collins Jennifer L. , Ringe Dagmar , Petsko Gregory A.
Published: February 2004

The yeast geneYDR533Cencodes a protein belonging to the DJ-1/ThiJ/PfpI superfamily. This family includes the human protein DJ-1, which is mutated in autosomal recessive early-onset Parkinson's disease. The function of DJ-1 and its yeast homologue YDR533Cp is unknown. We report here the crystal structure of YDR533Cp at 1.8-Å resolution. The structure indicates that the closest relative to YDR533Cp is theEscherichia coliheat shock protein Hsp31 (YedU), which has both chaperone and protease activity. As expected, the overall fold of the core domain of YDR533Cp is also similar to that of DJ-1 and the bacterial protease PfpI. YDR533Cp contains a possible catalytic triad analogous to that of Hsp31 and an additional domain that is present in Hsp31 but is not seen in DJ-1 and other members of the family. The cysteine in this triad (Cys-138) is oxidized in this crystal structure, similar to modifications seen in the corresponding cysteine in the crystal structure of DJ-1. YDR533Cp appears to be a dimer both in solution and the crystal, but this dimer is formed by a different interface than that found in Hsp31 or other members of the superfamily.
Histone H3.3 is enriched in covalent modifications associated with active chromatin
Authors: McKittrick Erin , Gafken Philip R. , Ahmad Kami , Henikoff Steven
Published: February 2004

Chromatin states can be distinguished by differential covalent modifications of histones or by utilization of histone variants. Chromatin associated with transcriptionally active loci becomes enriched for histones with particular lysine modifications and accumulates the H3.3 histone variant, the substrate for replication-independent nucleosome assembly. However, studies of modifications at particular loci have not distinguished between histone variants, so the relationship among modifications, histone variants, and nucleosome assembly pathways is unclear. To address this uncertainty, we have quantified the relative abundance of H3 and H3.3 and their lysine modifications. Using a
Drosophila
cell line system in which H3.3 has been shown to specifically package active loci, we found that H3.3 accounts for ≈25% of total histone 3 in bulk chromatin, enough to package essentially all actively transcribed genes. MS and antibody characterization of separated histone 3 fractions revealed that H3.3 is relatively enriched in modifications associated with transcriptional activity and deficient in dimethyl lysine-9, which is abundant in heterochromatin. To explain enrichment on alternative variants, we propose that histone modifications are tied to the alternative nucleosome assembly pathways that use primarily H3 at replication forks and H3.3 at actively transcribed genes in a replication-independent manner.
STAT1 is overexpressed in tumors selected for radioresistance and confers protection from radiation in transduced sensitive cells
Authors: Khodarev Nikolai N. , Beckett Michael , Labay Edwardine , Darga Thomas , Roizman Bernard , Weichselbaum Ralph R.
Published: February 2004

Nu61, a radiation-resistant human tumor xenograft, was selected from a parental radiosensitive tumor SCC-61 by eight serial cycles of passage in athymic nude mice and
in vivo
irradiation. Replicate DNA array experiments identified 52 genes differentially expressed in nu61 tumors compared with SCC-61 tumors. Of these, 19 genes were in the IFN-signaling pathway and moreover, 25 of the 52 genes were inducible by IFN in the nu61 cell line. Among the genes involved in IFN signaling, STAT1α and STAT1β were the most highly overexpressed in nu61 compared to SCC-61. STAT1α and STAT1β cDNAs were cloned and stably transfected into SCC-61 tumor cells. Clones of SCC-61 tumor cells transfected with vectors expressing STAT1α and STAT1β demonstrated radioprotection after exposure to 3 Gy (
P
< 0.038). The results indicate that radioresistance acquired during radiotherapy treatment may account for some treatment failures and demonstrate an association of acquired tumor radioresistance with up-regulation of components of the IFN-related signaling pathway.
The
Drosophila
gene
Start1
: A putative cholesterol transporter and key regulator of ecdysteroid synthesis
Authors: Roth Guenther E. , Gierl Mathias S. , Vollborn Lars , Meise Martin , Lintermann Ruth , Korge Guenter
Published: February 2004

Human metastatic lymph node 64 (MLN64) is a transmembrane protein that shares homology with the cholesterol-binding vertebrate steroid acute regulatory protein (StAR)-related lipid transfer domain (START) and is involved in cholesterol traffic and steroid synthesis. We identified a
Drosophila melanogaster
gene whose putative protein product shows extensive homology with MLN64 and that we name
Start1
(FlyBase CG3522). The putative Start1 protein, derived from
Start1
cDNA sequences, contains an additional 122 aa of unknown function within the StAR-related lipid transfer domain. Similar inserts seem to exist in the
Start1
homologues of
Drosophila pseudoobscura
and
Anopheles gambiae
, but not in the homologous protein of the urochordate
Ciona intestinalis
. Immunostaining using an insert-specific antibody confirms the presence of the insert in the cytoplasm. Whereas RT-PCR data indicate that
Start1
is expressed ubiquitously, RNA
in situ
hybridizations demonstrate its overexpression in prothoracic gland cells, where ecdysteroids are synthesized from cholesterol. Transcripts of
Start1
are detectable in embryonic ring gland progenitor cells and are abundant in prothoracic glands of larvae showing wave-like expression during larval stages. In adults,
Start1
is expressed in nurse cells of the ovary. These observations are consistent with the assumption that
Start1
plays a key role in the regulation of ecdysteroid synthesis. Vice versa, the expression of
Start1
itself seems to depend on ecdysone, as in the ecdysone-deficient mutant
ecd-1, Start1
expression is severely reduced.
T cell receptor ζ reconstitution fails to restore responses of T cells rendered hyporesponsive by tumor necrosis factor α
Authors: Clark Joanna M. , Annenkov Alexander E. , Panesar Manvinder , Isomäki Pia , Chernajovsky Yuti , Cope Andrew P.
Published: February 2004

Expression and function of the antigen T cell receptor (TCR) play a central role in regulating immune responsiveness. Accordingly, targeting the expression of TCRαβ or its associated CD3 subunits profoundly influences T cell development and adaptive immunity. Down-regulation of the invariant TCRζ chain has been documented in a wide variety of chronic inflammatory and infectious diseases, and is thought to contribute to the paradoxical immune suppression observed in these diseases. Previously, we reported that prolonged exposure of T cell hybridoma clones to tumor necrosis factor α (TNF) induces nondeletional and reversible hyporesponsiveness to TCR engagement, associated with down-regulation of TCRζ chain expression, impaired TCR/CD3 complex assembly, and attenuation of TCR-induced membrane proximal tyrosine phosphorylation. Here, we have tested whether receptor specific T cell responses are rescued in TNF-treated T cell hybridomas by retroviral-mediated expression of ζ-chimeric (C2ζ) receptors or wild-type TCRζ. Expression of C2ζ receptors at the cell surface is relatively refractory to chronic TNF stimulation. However, C2ζ receptor function depends on association with endogenous TCRζ chains, whose expression is down-regulated by TNF, and so C2 receptor specific responses are attenuated in TNF-treated T cells. Unexpectedly, overexpression of wild-type TCRζ maintains cell surface TCR/CD3 complex expression but fails to rescue receptor proximal signaling in TNF-treated T cells, suggesting the existence of hitherto unrecognized mechanisms through which TNF regulates T cell responsiveness. We provide additional evidence that TNF also uncouples distal TCR signaling pathways independently of its effects on TCRζ expression.
Elucidation of signaling properties of vasopressin receptor-related receptor 1 by using the chimeric receptor approach
Authors: Gupte Jamila , Cutler Gene , Chen Jin-Long , Tian Hui
Published: February 2004

The identification of endogenous or surrogate ligands for orphan G protein-coupled receptors (GPCRs) represents one of the most important tasks in GPCR biology and pharmacology. The challenge lies in choosing an appropriate assay in the absence of ways to activate the receptor of interest. We investigated the signaling pathway for an orphan GPCR referred to here as vasopressin receptor-related receptor 1 (VRR1) by generating a chimeric receptor, V1a/VRR1. The engineered construct contained vasopressin V1a receptor with all three intracellular loops and C terminus replaced by those of VRR1. The chimera behaved like a typical GPCR when transiently and stably expressed in mammalian cell lines based on radioligand binding and receptor internalization studies. Upon arginine vasopressin stimulation, this chimeric receptor induced robust calcium mobilization and increase of adenylate cyclase activity. The observed signaling activities are through the activation of the chimera instead of endogenously expressed receptors, as single amino acid changes in the second transmembrane regions of the chimera drastically reduced receptor efficacy and potency. Our results suggest that VRR1 has dual signaling properties in coupling to both G
q
and G
S
pathways. Analysis of native VRR1 receptor signaling pathway by using a recently identified ligand for VRR1 confirmed this conclusion and therefore validated the utility of the chimeric receptor approach for signaling pathway identification.
The gene for solubleN-ethylmaleimide sensitive factor attachment protein α is mutated in hydrocephaly with hop gait (hyh) mice
Authors: Hong Hee-Kyung , Chakravarti Aravinda , Takahashi Joseph S.
Published: February 2004

The spontaneous autosomal recessive mouse mutant for hydrocephaly with hop gait (hyh) exhibits dramatic cystic dilation of the ventricles at birth and invariably develops hopping gait. We show that the gene for solubleN-ethylmaleimide sensitive factor attachment protein α, also known as α-SNAP, is mutated inhyhmice. α-SNAP plays a key role in a wide variety of membrane fusion events in eukaryotic cells, including the regulated exocytosis of neurotransmitters. Homozygous mutant mice harbor a missense mutation M105I in a conserved residue in one of the α-helical domains. We demonstrate that thehyhmutant is not a null allele and is expressed; however, the mutant protein is 40% less abundant inhyhmice. Thehyhmutant provides a valuablein vivomodel to study vesicle/membrane trafficking and provides insight into the potential roles of α-SNAP in embryogenesis and brain development.
Genome annotation by high-throughput 5′ RNA end determination
Authors: Hwang Byung Joon , Müller Hans-Michael , Sternberg Paul W.
Published: February 2004

Complete gene identification and annotation, including alternative transcripts, remains a challenge in understanding genome organization. Such annotation can be achieved by a combination of computational analysis and experimental confirmation. Here, we describe a high-throughput technique, trans-spliced exon coupled RNA end determination (TEC-RED), that identifies 5′ ends of expressed genes in nematodes. TEC-RED can distinguish coding regions from regulatory regions and identify genes as well as their alternative transcripts that have different 5′ ends. Application of TEC-RED to ≈10% of the
Caenorhabditis elegans
genome yielded tags 75% of which experimentally verified predicted 5′-RNA ends and 25% of which provided previously unknown information about 5′-RNA ends, including the identification of 99 previously unknown genes and 32 previously unknown operons. This technique will be applicable in any organisms that have a trans-splicing reaction from spliced leader RNA. We also describe an efficient sequential method for concatenating short sequence tags for any serial analysis of gene expression-like techniques.
Cell-cell signaling controlsXylella fastidiosainteractions with both insects and plants
Authors: Newman Karyn L. , Almeida Rodrigo P. P. , Purcell Alexander H. , Lindow Steven E.
Published: February 2004

Xylella fastidiosa, which causes Pierce's disease of grapevine and other important plant diseases, is a xylem-limited bacterium that depends on insect vectors for transmission. Although many studies have addressed disease symptom development and transmission of the pathogen by vectors, little is known about the bacterial mechanisms driving these processes. Recently availableX. fastidiosagenomic sequences and molecular tools have provided new routes for investigation. Here, we show that a diffusible signal molecule is required for biofilm formation in the vector and for vector transmission to plants. We constructed strains ofX. fastidiosamutated in therpfFgene and determined that they are unable to produce the signal activity. In addition,rpfFmutants are more virulent than the wild type when mechanically inoculated into plants. This signal therefore directs interaction ofX. fastidiosawith both its insect vector and plant host. Interestingly,rpfFmutants can still formin plantabiofilms, which differ architecturally from biofilms in insects, suggesting that biofilm architecture, rather than a passive response to the environment, is actively determined byX. fastidiosagene expression. This article reports a cell-cell signaling requirement for vector transmission. Identification of the genes regulated byrpfFshould elucidate bacterial factors involved in transmission and biofilm formation in the insect.
Divergent gene copies in the asexual class Bdelloidea (Rotifera) separated before the bdelloid radiation or within bdelloid families
Authors: Welch David B. Mark , Cummings Michael P. , Hillis David M. , Meselson Matthew
Published: February 2004

Rotifers of the asexual class Bdelloidea are unusual in possessing two or more divergent copies of every gene that has been examined. Phylogenetic analysis of the heat-shock genehsp82and the TATA-box-binding protein genetbpin multiple bdelloid species suggested that for each gene, each copy belonged to one of two lineages that began to diverge before the bdelloid radiation. Such gene trees are consistent with the two lineages having descended from former alleles that began to diverge after meiotic segregation ceased or from subgenomes of an alloploid ancestor of the bdelloids. However, the original analyses of bdelloid gene-copy divergence used only a single outgroup species and were based on parsimony and neighbor joining. We have now used maximum likelihood and Bayesian inference methods and, forhsp82, multiple outgroups in an attempt to produce more robust gene trees. Here we report that the available data do not unambiguously discriminate between gene trees that root the origin ofhsp82andtbpcopy divergence before the bdelloid radiation and those which indicate that the gene copies began to diverge within bdelloid families. The remarkable presence of multiple diverged gene copies in individual genomes is nevertheless consistent with the loss of sex in an ancient ancestor of bdelloids.
RETRACTED: Molecular dissection of the roles of nucleotide binding and hydrolysis in dynein's AAA domains in
Saccharomyces cerevisiae
Authors: Reck-Peterson Samara L. , Vale Ronald D.
Published: February 2004

The motor protein cytoplasmic dynein is responsible for most of the minus-end-directed microtubule traffic within cells. Dynein contains four evolutionarily conserved AAA (ATPase associated with various cellular activities) domains that are thought to bind nucleotide; the role of nucleotide binding and hydrolysis in each of these four AAA domains has constituted an important and unresolved question in understanding dynein's mechanism. Using
Saccharomyces cerevisiae
cytoplasmic dynein as a model system, we mutagenized residues involved in nucleotide binding or hydrolysis in the four AAA domains and examined the ability of the mutant dyneins to mediate nuclear segregation
in vivo
and to bind microtubules
in vitro
. Our analysis shows that an AAA1 hydrolysis mutant blocks dynein function, whereas a triple AAA2/3/4 hydrolysis mutant does not, suggesting that nucleotide binding is required at only one site. We also show that nucleotide binding at AAA3, but not hydrolysis, is essential for motor activity
in vivo
and ATP-induced dissociation of dynein from microtubules, suggesting that this domain acts as a critical allosteric site. In contrast, mutations in AAA2 cause subtle defects in dynein function, whereas mutation in AAA4 produce no obvious defects. These results show that the four conserved dynein AAA domains have distinct functions in dynein's mechanochemical cycle.
A model of magnesium metabolism in young sheep: transactions between plasma, cerebrospinal fluid and bone
Authors: Robson A. B. , Sykes A. R. , McKinnon A. E. , Bell S. T.
Published: February 2004

An extension of the previously proposed model of Mg metabolism (Robson et al. 1997) has been developed to consider the transactions of Mg that are associated with cerebrospinal fluid (CSF) and bone. The representation of the CSF as a single Mg compartment with uptake from the plasma described by Michaelis–Menten kinetics gives very good agreement with published experiments. Analysis of the available information on resorption of Mg from adult bone indicates that this process makes a negligible contribution to Mg homeostasis and can be omitted from the model.
Diet, nutrition and the prevention of excess weight gain and obesity
Authors: BA Swinburn , I Caterson , JC Seidell , WPT James
Published: February 2004

Objective:

To review the evidence on the diet and nutrition causes of obesity and to recommend strategies to reduce obesity prevalence.

Design:

The evidence for potential aetiological factors and strategies to reduce obesity prevalence was reviewed, and recommendations for public health action, population nutrition goals and further research were made.

Results:

Protective factors against obesity were considered to be: regular physical activity (convincing); a high intake of dietary non-starch polysaccharides (NSP)/fibre (convincing); supportive home and school environments for children (probable); and breastfeeding (probable). Risk factors for obesity were considered to be sedentary lifestyles (convincing); a high intake of energy-dense, micronutrient-poor foods (convincing); heavy marketing of energy-dense foods and fast food outlets (probable); sugar-sweetened soft drinks and fruit juices (probable); adverse social and economic conditions—developed countries, especially in women (probable).

A broad range of strategies were recommended to reduce obesity prevalence including: influencing the food supply to make healthy choices easier; reducing the marketing of energy dense foods and beverages to children; influencing urban environments and transport systems to promote physical activity; developing community-wide programmes in multiple settings; increased communications about healthy eating and physical activity; and improved health services to promote breastfeeding and manage currently overweight or obese people.

Conclusions:

The increasing prevalence of obesity is a major health threat in both low- and high income countries. Comprehensive programmes will be needed to turn the epidemic around.
Diet, nutrition and the prevention of hypertension and cardiovascular diseases
Authors: Reddy K Srinath , Katan Martijn B
Published: February 2004

Cardiovascular diseases (CVD) are growing contributors to global disease burdens, with epidemics of CVD advancing across many regions of the world which are experiencing a rapid health transition. Diet and nutrition have been extensively investigated as risk factors for major cardiovascular diseases like coronary heart disease (CHD) and stroke and are also linked to other cardiovascular risk factors like diabetes, high blood pressure and obesity. The interpretation of evidence needs to involve a critical appraisal of methodological issues related to measurement of exposures, nature of outcome variables, types of research design and careful separation of cause, consequence and confounding as the basis for observed associations.

Adequate evidence is available, from studies conducted within and across populations, to link several nutrients, minerals, food groups and dietary patterns with an increased or decreased risk of CVD. Dietary fats associated with an increased risk of CHD includetrans–fats and saturated fats, while polyunsaturated fats are known to be protective. Dietary sodium is associated with elevation of blood pressure, while dietary potassium lowers the risk of hypertension and stroke. Regular frequent intake of fruits and vegetables is protective against hypertension, CHD and stroke. Composite diets (such as DASH diets, Mediterranean diet, ‘prudent’ diet) have been demonstrated to reduce the risk of hypertension and CHD. Sufficient knowledge exists to recommend nutritional interventions, at both population and individual levels, to reduce cardiovascular risk. That knowledge should now be translated into policies which promote healthy diets and discourage unhealthy diets. This requires coordinated action at the level of governments, international organizations, civil society and responsible sections of the food industry.