bluuebunny/crossref_metadata_2025 huggingface dataset:
Yield and grain protein of wheat following phased perennial grass, lucerne, and annual pastures
Authors: Dear B. S. , Sandral G. A. , Virgona J. M. , Swan A. D.
Published: August 2004

The effect of using 4 perennial grasses or lucerne (Medicago sativa L.) in the pasture phase on subsequent wheat grain yield, protein, and grain hardness was investigated at 2 sites (Kamarah and Junee) in the south-eastern Australian cereal belt. The 6 perennial treatments were 5 mixtures of subterranean clover (Trifolium subterraneum L.), with one of lucerne, phalaris (Phalaris aquatica L.), cocksfoot (Dactylis glomerata L), wallaby grass (Austrodanthonia richardsonii (Cashm.) H.P. Linder), or lovegrass (Eragrostis curvula (Schrader) Nees cv. Consol), or one mixture of cocksfoot, phalaris, and lucerne. The results were compared with wheat after one of 3 annual pastures consisting of either pure subterranean clover, subterranean clover with annual volunteer broadleaf and grass weeds, or yellow serradella (Ornithopus compressus L.). The duration of the pasture phase was 3 years at the drier Kamarah site (av. annual rainfall 430 mm) and 4 years at Junee (550 mm). The effect of time of removal of the pastures in the year prior to cropping (28 August–3 September or 6–7 November) and the effect of nitrogen (N) fertiliser application were also examined.
In the absence of applied N, wheat grain yields at Kamarah were highest (4.7–4.9 t/ha) and grain protein lowest (10.3–11.1%) following phalaris, wallaby grass, and cocksfoot. Grain protein levels were highest (12.9–13.9%) in wheat following the 3 annual legume swards at both sites. Previous pasture type had no effect on wheat yields at the Junee site. Wheat grain protein and total N taken up by the crop were positively related to available soil N to 100 cm measured at sowing at both sites. Grain protein was inversely related to grain yield at both sites where additional N fertiliser was added, but not in the absence of fertiliser N. There was a positive response in grain protein to delayed time of pasture removal in second year wheat at Junee. The application of additional N fertiliser increased grain protein of wheat following all 9 pasture types at the drier Kamarah site, but at the Junee site there was only a positive grain protein response following phalaris, cocksfoot, and wallaby grass.
Early removal of the pasture prior to cropping increased soil water (10–130 cm) at sowing by 18 mm, delayed wheat senescence, and increased crop yield by 11% (0.44 t/ha) at the drier Kamarah site. Early removal of the pasture at Junee increased soil water by 29 mm, crop yields by 2% (0.14 t/ha), and increased grain protein in wheat following cocksfoot, wallaby grass, and phalaris, but not following the 3 annual legume treatments.
The study demonstrated that perennial grasses can be successfully incorporated into phased rotations with wheat without affecting grain yield, but protein levels may be lower and timing of pasture removal will be important to limit the effect of water deficits on grain yield.
Incidence and effects of viruses on production of two newly adopted hop (Humulus lupulus) cultivars in Australia
Authors: Pethybridge S. J. , Wilson C. R. , Leggett G. W.
Published: August 2004

The incidence and spread of Hop latent virus (HpLV), Hop mosaic virus (HpMV), and Apple mosaic virus (ApMV) in nurseries and gardens established with elite material (certified free of HpLV, HpMV, and ApMV by serological testing), and the effect of these viruses on production of 2 newly adopted hop (Humulus lupulus) cultivars were assessed in Tasmania, Australia. Infection by HpLV in Agate was associated with significant losses in dry cone yield of 40–70% compared with the yield of elite plants. Infection by HpLV in Agate was also associated with a 44% reduction in alpha acid levels and the cohumulone proportion of the alpha acids. However, in the second year, no significant reductions in levels of alpha acid and beta acid contents, or in the ratio of alpha to beta acids were detected. Plants infected by HpLV were also significantly shorter than elite plants in both seasons. No significant effects on growth or yield were detected for HpMV in Agate or for any of the virus combinations tested in Super Pride. The incidences and spread of HpLV, HpMV, and ApMV were consistently higher in Agate than in Super Pride and the incidences of all these viruses were higher at Forrester River than at Bushy Park and Gunns Plains. The presence of viruses in nurseries containing material propagated from elite mother plants is also of concern and may be contributing to high levels of virus incidence found soon after establishment. When combined, this information can be used to design control strategies for the Australian hop industry for these cultivars.
Genetic analysis of coleoptile length and diameter in wheat
Authors: Rebetzke G. J. , Richards R. A. , Sirault X. R. R. , Morrison A. D.
Published: August 2004

Lack of moisture near the soil surface commonly delays sowing, reducing grain yields of Australian wheat (Triticum aestivum) crops. Deep sowing would allow growers to make use of soil moisture lying below the drying topsoil, but the short coleoptiles of semidwarf wheats reduce emergence when sowing at depths greater than 5 cm. Selection of longer, thicker coleoptiles would help in improving emergence in hard or crusted soils, or when deep sowing, yet little is known of genetic control of coleoptile size in wheat. A diallel mating design was generated from crosses between 12 Australian and overseas wheats, and assessed for coleoptile size at different temperatures (11, 15, 19, and 23°C). Repeatabilities for coleoptile diameter and length were moderate to high on an entry-mean basis (R2 = 0.48 and 0.77, respectively), reflecting large genotype and small genotype × temperature interaction variances. Genotypic variation among parents translated into large and significant (P < 0.01) differences among F1 progeny (94–142 mm and 1.56–1.84 mm for length and diameter, respectively). General (GCA) and specific combining ability (SCA), and reciprocal effects were significant (P < 0.01) for length and diameter. Baker’s GCA/SCA ratio was high (0.62–0.77) for coleoptile length but intermediate for diameter (0.38–0.64), indicating strong additive genetic control for length. Further, GCA effects and parental means were strongly correlated (r = 0.81–0.91, P < 0.01) indicating parent length to be a useful predictor of progeny performance. Coleoptile lengths for progeny derived from Rht8, Rht9, and Rht12 dwarfing gene donors were generally shorter (c. –7 to –13%) but were still an average 47% longer than coleoptiles of Rht-B1b and Rht-D1b controls. The genetic correlation for coleoptile length and diameter was small (rg = –0.25 ± 0.15n.s.) suggesting that the two traits are genetically independent. Development of wheats with longer, thicker coleoptiles should be readily achieved in selection among partially inbred families from crosses targetting improved establishment.
The mouse kinome: Discovery and comparative genomics of all mouse protein kinases
Authors: Caenepeel Sean , Charydczak Glen , Sudarsanam Sucha , Hunter Tony , Manning Gerard
Published: August 2004

We have determined the full protein kinase (PK) complement (kinome) of mouse. This set of 540 genes includes many novel kinases and corrections or extensions to >150 published sequences. The mouse has orthologs for 510 of the 518 human PKs. Nonorthologous kinases arise only by retrotransposition and gene decay. Orthologous kinase pairs vary in sequence conservation along their length, creating a map of functionally important regions for every kinase pair. Many species-specific sequence inserts exist and are frequently alternatively spliced, allowing for the creation of evolutionary lineage-specific functions. Ninety-seven kinase pseudogenes were found, all distinct from the 107 human kinase pseudogenes. Chromosomal mapping links 163 kinases to mutant phenotypes and unlocks the use of mouse genetics to determine functions of orthologous human kinases.
In different organisms, the mode of interaction between two signaling proteins is not necessarily conserved
Authors: Park Sang-Youn , Beel Bryan D. , Simon Melvin I. , Bilwes Alexandrine M. , Crane Brian R.
Published: August 2004

Although interfaces mediating protein–protein interactions are thought to be under strong evolutionary constraints, binding of the chemotaxis histidine kinase CheA to its phosphorylation target CheY suggests otherwise. The structure ofThermotoga maritimaCheA domain P2 in complex with CheY reveals a different association than that observed for the sameEscherichia coliproteins. Similar regions of CheY bind CheA P2 in the two systems, but the CheA P2 domains differ by an ≈90° rotation. CheA binds CheY with identical affinity inT. maritimaandE. coliat the vastly different temperatures where the respective organisms live. Distinct sets of P2 residues mediate CheY binding in the two complexes; conservation patterns of these residues in CheA and compensations in CheY delineate two families of prokaryotic chemotaxis systems. A protein complex that has the same components and general function in different organisms, but an altered structure, indicates unanticipated complexity in the evolution of protein–protein interactions and cautions against extrapolating structural data from homologs.
The mouse
juvenile spermatogonial depletion
(
jsd
) phenotype is due to a mutation in the X-derived retrogene,
mUtp14b
Authors: Rohozinski Jan , Bishop Colin E.
Published: August 2004

The recessive juvenile spermatogonial depletion (
jsd
) mutation results in a single wave of spermatogenesis, followed by failure of type A spermatogonia to differentiate, resulting in adult male sterility. We have identified a
jsd
-specific rearrangement in the mouse homologue of the
Saccharomyces cerevisiae
gene
UTP14
, termed
mUtp14b
. Confirmation that
mUtp14b
underlies the
jsd
phenotype was obtained by transgenic bacterial artificial chromosome (BAC) rescue. We also identified a homologous gene on the
Mus musculus
X chromosome (MMUX) (
mUtp14a
) that is the strict homologue of the yeast gene, from which the intronless
mUtp14b
has been derived by retrotransposition. Expression analysis showed that
mUtp14b
is predominantly expressed in the germ line of the testis from zygotene through round spermatids, whereas
mUtp14a
, although well expressed in all somatic tissues, could be detected only in the germ line in round spermatids. In yeast, depletion of the UTP proteins impedes production of 18S rRNA, leading to cell death. We propose that the retroposed autosomal copy
mUtp14b
, having acquired a testis-specific expression pattern, could have provided a mechanism for increasing the efficiency and/or numbers of germ cells produced by meeting the need for more 18S rRNA and protein. Such a mechanism would be of obvious reproductive advantage and be strongly selected for in evolution. Consistent with this hypothesis is the finding of a similar X-autosome retroposition of UTP14 in human which seems to have arisen independently of that in rodents. In
jsd
homozygotes, which lack a functional copy of
Utp14b
, insufficient production of rRNA quickly leads to a cessation of spermatogenesis.
Advanced glycation endproduct (AGE) receptor 1 is a negative regulator of the inflammatory response to AGE in mesangial cells
Authors: Lu Changyong , He John Cijiang , Cai Weijing , Liu Huixian , Zhu Li , Vlassara Helen
Published: August 2004

Advanced glycation endproducts (AGE) contribute to kidney disease due to diabetes or aging by means of mesangial cell (MC) receptors, such as the receptor for AGE (RAGE), which promote oxidant-stress-dependent NF-κB activation and inflammatory gene expression. MC also express scavenger receptors SR-I and SR-II and AGE receptors 1, 2, and 3 (AGE-R1, -R2, and -R3), some of which are linked to AGE turnover. Because AGE-R1 expression is found suppressed in severe diabetic kidney disease, as other receptors increase, we investigated whether his molecule has a protective role against AGE-induced MC injury. A stable murine MC line overexpressing AGE-R1 (R1-MC) was generated, exhibiting a 1.8- to 2.7-fold increase in
125
I-AGE-specific binding, uptake, and degradation, compared with mock-MC. However, AGE-stimulated NF-κB activity and mitogen-activated protein kinase (MAPK) (p44/42) phosphorylation were found markedly suppressed in R1-MC. Additionally, AGE-stimulated macrophage chemotaxis protein 1 and RAGE overexpression were abolished in R1-MC. The effect of R1 on RAGE signaling was investigated after overexpressing RAGE in Chinese hamster ovary cells, which lack RAGE. AGE stimulation elicited NF-κB and MAPK activities in RAGE-Chinese hamster ovary cells; however, after cotransfection with R1, these responses were suppressed. Also, after silencing endogenous R1 in wild-type MC by R1 small interfering RNA, AGE-mediated MAPK/p44/42 activation exceeded by >2-fold that of mock-MC, consistent with loss of the activation-inhibitory properties of native AGE-R1. AGE-R1, although enhancing AGE removal, is also a distinct receptor in that it suppresses AGE-mediated MC inflammatory injury through negative regulation of RAGE, a previously uncharacterized pathway that may protect renal and other tissue injury due to diabetes and aging.
High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of splitRenillaluciferase
Authors: Kim Sung Bae , Ozawa Takeaki , Watanabe Shigeaki , Umezawa Yoshio
Published: August 2004

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteinsin vitroandin vivo. The principle is based on reconstitution of split fragments ofRenilla reniformis(Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-basedin vitrosensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasivein vivoimaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analysesin vitroandin vivoprovide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.
Lightoid and Claret: A rab GTPase and its putative guanine nucleotide exchange factor in biogenesis of
Drosophila
eye pigment granules
Authors: Ma Jinping , Plesken Heide , Treisman Jessica E. , Edelman-Novemsky Irit , Ren Mindong
Published: August 2004

To elucidate the biogenetic pathways for the generation of lysosome-related organelles, we have chosen to study the
Drosophila
eye pigment granules because they are lysosome-related and the fruit fly provides the advantages of a genetic system in which many mutations affect eye color. Here, we report the molecular identification of two classic
Drosophila
eye-color genes required for pigment granule biogenesis,
claret
and
lightoid
; the former encodes a protein containing seven repeats with sequence similarity to those that characterize regulator of chromosome condensation 1 (RCC1, a guanine nucleotide exchange factor for the small GTPase, Ran), and the latter encodes a rab GTPase, Rab-RP1. We demonstrate in transfected cells that Claret, through its RCC1-like domain, interacts preferentially with the nucleotide-free form of Rab-RP1, and this interaction involves Claret's first three RCC1-like repeats that are also critical for Claret's function in pigment granule biogenesis in transgenic rescue experiments. In addition, double-mutant analyses suggest that the gene products of
claret
and
lightoid
function in the same pathway, which is different from that of
garnet
and
ruby
(which encode the δ- and β-subunit of the tetrameric adaptor protein 3 complex, respectively). Taken together, our results suggest that Claret functions as a guanine nucleotide exchange factor for Lightoid/Rab-RP1 in an adaptor protein 3-independent vesicular trafficking pathway of pigment granule biogenesis.
Rhodopsin kinase activity modulates the amplitude of the visual response in
Drosophila
Authors: Lee Seung-Jae , Xu Hong , Montell Craig
Published: August 2004

A feature shared between
Drosophila
rhodopsin and nearly all other G protein-coupled receptors is agonist-dependent protein phosphorylation. Despite extensive analyses of
Drosophila
phototransduction, the identity and function of the rhodopsin kinase (RK) have been elusive. Here, we provide evidence that G protein-coupled receptor kinase 1 (GPRK1), which is most similar to the β-adrenergic receptor kinases, G protein-coupled receptor kinase 2 (GRK2) and GRK3, is the fly RK. We show that GPRK1 is enriched in photoreceptor cells, associates with the major
Drosophila
rhodopsin, Rh1, and phosphorylates the receptor. As is the case with mammalian GRK2 and GRK3,
Drosophila
GPRK1 includes a C-terminal pleckstrin homology domain, which binds to phosphoinositides and the Gβγ subunit. To address the role of GPRK1, we generated transgenic flies that expressed higher and lower levels of RK activity. Those flies with depressed levels of RK activity displayed a light response with a much larger amplitude than WT. Conversely, the amplitude of the light response was greatly suppressed in transgenic flies expressing abnormally high levels of RK activity. These data point to an evolutionarily conserved role for GPRK1 in modulating the amplitude of the visual response.
A single-amino-acid lid renders a gas-tight compartment within a membrane-bound transporter
Authors: Salomonsson Lina , Lee Alex , Gennis Robert B. , Brzezinski Peter
Published: August 2004

Proteins undergo structural fluctuations between nearly isoenergetic substates. Such fluctuations are often intimately linked with the functional properties of proteins. However, in some cases, such as in transmembrane ion transporters, the control of the ion transport requires that the protein is designed to restrict the motions in specific regions. In this study, we have investigated the dynamics of a membrane-bound respiratory oxidase, which acts both as an enzyme catalyzing reduction of O
2
to H
2
O and as a transmembrane proton pump. The segment of the protein where proton translocation is controlled (“gating” region) overlaps with a channel through which O
2
is delivered to the catalytic site. We show that the replacement of an amino acid residue with a small side chain (Gly) by one with a larger side chain (Val), in a narrow part of this channel, completely blocks the O
2
access to the catalytic site and results in formation of a compartment around the site that is impermeable to small gas molecules. Thus, the protein motions cannot counter the blockage introduced by the mutation. These results indicate that the protein motions are restricted in the proton-gating region and that rapid O
2
delivery to the catalytic site requires a gas channel, which is confined within a rigid protein body.
Breed distribution and history of canine
mdr1-1Δ
, a pharmacogenetic mutation that marks the emergence of breeds from the collie lineage
Authors: Neff Mark W. , Robertson Kathryn R. , Wong Aaron K. , Safra Noa , Broman Karl W. , Slatkin Montgomery , Mealey Katrina L. , Pedersen Niels C.
Published: August 2004

A mutation in the canine multidrug resistance gene,
MDR1
, has previously been associated with drug sensitivities in two breeds from the collie lineage. We exploited breed phylogeny and reports of drug sensitivity to survey other purebred populations that might be genetically at risk. We found that the same allele,
mdr1-1
Δ, segregated in seven additional breeds, including two sighthounds that were not expected to share collie ancestry. A mutant haplotype that was conserved among affected breeds indicated that the allele was identical by descent. Based on breed histories and the extent of linkage disequilibrium, we conclude that all dogs carrying
mdr1-1
Δ are descendants of a dog that lived in Great Britain before the genetic isolation of breeds by registry (
ca.
1873). The breed distribution and frequency of
mdr1-1
Δ have applications in veterinary medicine and selective breeding, whereas the allele's history recounts the emergence of formally recognized breeds from an admixed population of working sheepdogs.
Crystal structure of
Caenorhabditis elegans
HER-1 and characterization of the interaction between HER-1 and TRA-2A
Authors: Hamaoka Brent Y. , Dann Charles E. , Geisbrecht Brian V. , Leahy Daniel J.
Published: August 2004

HER-1 is a secreted protein that promotes male development in the nematode
Caenorhabditis elegans
. HER-1 inhibits the function of TRA-2A, a multipass integral membrane protein thought to serve as its receptor. We report here the 1.5-Å crystal structure of HER-1. The structure was solved by the multiwavelength anomalous diffraction method by using selenomethionyl-substituted HER-1 produced in Chinese hamster ovary cells. The HER-1 structure consists of two all-helical domains and is not closely homologous to any known structure. Sites of amino acid substitutions known to impair HER-1 function were mapped on the HER-1 structure and classified according to the likely mechanism by which they affect HER-1 activity. A subset of these and other amino acid substitutions on the HER-1 surface were assayed for their ability to disrupt interactions between HER-1 and TRA-2A-expressing cells, and a localized region on the HER-1 surface important for mediating this interaction was identified.
The structure of human parvovirus B19
Authors: Kaufmann Bärbel , Simpson Alan A. , Rossmann Michael G.
Published: August 2004

Human parvovirus B19 is the only parvovirus known to be a human pathogen. The structure of recombinant B19-like particles has been determined to ≈3.5-Å resolution by x-ray crystallography and, to our knowledge, represents the first near-atomic structure of an
Erythrovirus
. The polypeptide fold of the major capsid protein VP2 is a “jelly roll” with a β-barrel motif similar to that found in many icosahedral viruses. The large loops connecting the strands of the β-barrel form surface features that differentiate B19 from other parvoviruses. Although B19 VP2 has only 26% sequence identity to VP3 of adeno-associated virus, 72% of the C
α
atoms can be aligned structurally with a rms deviation of 1.8 Å. Both viruses require an integrin as a coreceptor, and conserved surface features suggest a common receptor-binding region.
Coactivator AIB1 links estrogen receptor transcriptional activity and stability
Authors: Shao Wenlin , Keeton Erika Krasnickas , McDonnell Donald P. , Brown Myles
Published: August 2004

Agonist-mediated degradation of estrogen receptor α (ERα) has been associated with its transcriptional activity. However, the mechanism by which ERα is targeted for degradation and whether there is a direct functional link between ERα stability and ERα-mediated transactivation have not been elucidated. Here we provide evidence that the p160 coactivator, AIB1, uniquely mediates agonist-induced, but not antagonist-induced, ERα degradation. We show that AIB1 recruitment by ERα is not only necessary but also sufficient to promote degradation. Suppression of AIB1 levels leads to ERα stabilization in the presence of 17β-estradiol and, despite increased ERα levels, reduced recruitment of ERα to endogenous target gene promoters. In addition, association of RNA polymerase II with ERα target promoters is lost when AIB1 is suppressed, leading to inhibition of target gene transcription. AIB1 thus plays a dual role in regulating ERα activity, one in recruiting transcription factors including other coactivators involved in gene activation and the other in regulating ERα protein degradation mediated by the ubiquitin–proteosome machinery.
Genes encoding candidate pheromone receptors in a moth (
Heliothis virescens
)
Authors: Krieger J. , Grosse-Wilde E. , Gohl T. , Dewer Y. M. E. , Raming K. , Breer H.
Published: August 2004

The remarkable responsiveness of male moths to female released pheromones is based on the extremely sensitive and selective reaction of highly specialized sensory cells in the male antennae. These cells are supposed to be equipped with male-specific receptors for pheromonal compounds, however, the nature of these receptors is still elusive. By using a combination of genomic sequence analysis and cDNA-library screening, we have cloned various cDNAs of the tobacco budworm
Heliothis virescens
encoding candidate olfactory receptors. A comparison of all identified receptor types not only highlighted their overall high degree of sequence diversity but also led to the identification of a small group of receptors sharing >40% identity. In RT-PCR analysis it was found that distinct members of this group were expressed exclusively in the antennae of male moths.
In situ
hybridization experiments revealed that the male-specific expression of these receptor types was confined to antennal cells located beneath sensillar hair structures (sensilla triochoidea), which have been shown to contain pheromone-sensitive neurons. Moreover, two-color double
in situ
-hybridization approaches uncovered that cells expressing one of these receptor types were surrounded by cells expressing pheromone-binding proteins, as expected for a pheromone-sensitive sensillum. These findings suggest that receptors like
Heliothis
receptor 14–16 (HR14–HR16) may render antennal cells responsive to pheromones.
The essence of total synthesis
Authors: Nicolaou K. C. , Snyder Scott A.
Published: August 2004

For the past century, the total synthesis of natural products has served as the flagship of chemical synthesis and the principal driving force for discovering new chemical reactivity, evaluating physical organic theories, testing the power of existing synthetic methods, and enabling biology and medicine. This perspective article seeks to examine this time-honored and highly demanding art, distilling its essence in an effort to ascertain its power and future potential.
Scaling theory for quasibrittle structural failure
Authors: Bažant Zdeněk P.
Published: August 2004

This inaugural article has a twofold purpose: (
i
) to present a simpler and more general justification of the fundamental scaling laws of quasibrittle fracture, bridging the asymptotic behaviors of plasticity, linear elastic fracture mechanics, and Weibull statistical theory of brittle failure, and (
ii
) to give a broad but succinct overview of various applications and ramifications covering many fields, many kinds of quasibrittle materials, and many scales (from 10
–8
to 10
6
m). The justification rests on developing a method to combine dimensional analysis of cohesive fracture with second-order accurate asymptotic matching. This method exploits the recently established general asymptotic properties of the cohesive crack model and nonlocal Weibull statistical model. The key idea is to select the dimensionless variables in such a way that, in each asymptotic case, all of them vanish except one. The minimal nature of the hypotheses made explains the surprisingly broad applicability of the scaling laws.
In vitro
transcription system delineates the distinct roles of the coactivators pCAF and p300 during MyoD/E47-dependent transactivation
Authors: Dilworth F. Jeffrey , Seaver Karen J. , Fishburn Anna L. , Htet Steve L. , Tapscott Stephen J.
Published: August 2004

The transcriptional coactivators p300 and pCAF are necessary for the myogenic factor MyoD to initiate the expression of skeletal muscle genes. In addition to mediating histone acetylation, both of these factors can acetylate MyoD; however, the complexity of cellular systems used to study MyoD has impeded delineation of the specific roles of these two acetyltransferases. Therefore, we established a MyoD-dependent
in vitro
transcription system that permits us to determine the roles of p300 and pCAF during MyoD-dependent transcriptional activation. Consistent with results from cellular systems, we demonstrate that maximal levels of transactivation
in vitro
require both p300 and pCAF, as well as the cofactor acetyl CoA. Dissection of the steps leading to transcription initiation revealed that the activities of p300 and pCAF are not redundant. During the initial stages of transactivation, p300 acetylates histone H3 and H4 within the promoter region and then recruits pCAF to MyoD. Once tethered to the promoter, pCAF acetylates MyoD to facilitate the transactivation process. Thus, we have established that pCAF and p300 carry out sequential and functionally distinct events on a promoter leading to transcriptional activation. Further dissection of this
in vitro
transcription system should be highly useful toward elucidating the mechanism by which coactivators facilitate differential gene expression by MyoD.
Dose-dependent response of FGF-2 for lymphangiogenesis
Authors: Chang Lynn K. , Garcia-Cardeña Guillermo , Farnebo Filip , Fannon Michael , Chen Emy J. , Butterfield Catherine , Moses Marsha A. , Mulligan Richard C. , Folkman Judah , Kaipainen Arja
Published: August 2004

Spatio-temporal studies on the growth of capillary blood vessels and capillary lymphatic vessels in tissue remodeling have suggested that lymphangiogenesis is angiogenesis-dependent. We revisited this concept by using fibroblast growth factor 2 (FGF-2) (80 ng) to stimulate the growth of both vessel types in the mouse cornea. When we lowered the dose of FGF-2 in the cornea 6.4-fold (12.5 ng), the primary response was lymphangiogenic. Further investigation revealed that vascular endothelial growth factor-C and -D are required for this apparent lymphangiogenic property of FGF-2, and when the small amount of accompanying angiogenesis was completely suppressed, lymphangiogenesis remained unaffected. Our findings demonstrate that there is a dose-dependent response of FGF-2 for lymphangiogenesis, and lymphangiogenesis can occur in the absence of a preexisting or developing vascular bed, i.e., in the absence of angiogenesis, in the mouse cornea.
Evolutionary genomics of ecological specialization
Authors: Zhong Shaobin , Khodursky Arkady , Dykhuizen Daniel E. , Dean Antony M.
Published: August 2004

We used a combination of genomic techniques to monitor chromosomal evolution across hundreds of generations as
Escherichia coli
adapted to growth-limiting concentrations of either lactulose, methyl-galactoside, or a 72:28 mixture of the two. DNA microarrays identified 8 unique duplications and 16 unique deletions among 42 evolvants from 23 chemostat experiments. Each mutation was confirmed by sequencing PCR-amplified flanking genomic DNA and, except for one deletion, an insertion sequence was found at the break point. vPCR of insertion sequences identified these same mutations and 16 additional insertions (all confirmed by sequencing). The pattern of genomic evolution is highly reproducible. Statistical analyses show that duplications at
lac
and mutations in
mgl
are adaptations specific to lactulose and to methyl-galactoside, respectively. Adaptation to mixed sugars is characterized by similar mutations, but
lac
duplications and
mgl
mutations usually arise in different backgrounds, producing ecological specialists for each sugar. This suggests that an antagonistic pleiotropic tradeoff between duplications at
lac
and mutations in
mgl
retards the evolution of generalists. Other mutations that repeatedly appear in replicate experiments are adaptations to the chemostat environment and are not specific to one or the other sugar.
Design for an optical cw atom laser
Authors: Ashkin Arthur
Published: August 2004

A new type of optical cw atom laser design is proposed that should operate at high intensity and high coherence and possibly record low temperatures. It is based on an “optical-shepherd” technique, in which far-off-resonance blue-detuned swept sheet laser beams are used to make new types of high-density traps, atom waveguides, and other components for achieving very efficient Bose–Einstein condensation and cw atom laser operation. A shepherd-enhanced trap is proposed that should be superior to conventional magneto-optic traps for the initial collection of molasses-cooled atoms. A type of dark-spot optical trap is devised that can cool large numbers of atoms to polarization-gradient temperatures at densities limited only by three-body collisional loss. A scheme is designed to use shepherd beams to capture and recycle essentially all of the escaped atoms in evaporative cooling, thereby increasing the condensate output by several orders of magnitude. Condensate atoms are stored in a shepherd trap, protected from absorbing light, under effectively zero-gravity conditions, and coupled out directly into an optical waveguide. Many experiments and devices may be possible with this cw atom laser.
Targeting cell division: Small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality
Authors: Margalit Danielle N. , Romberg Laura , Mets Rebecca B. , Hebert Alan M. , Mitchison Timothy J. , Kirschner Marc W. , RayChaudhuri Debabrata
Published: August 2004

FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis. We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ. Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association. These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively. We also show that Zantrins perturb FtsZ ring assembly in
Escherichia coli
cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents. Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target.
Marek's disease virus-encoded Meq gene is involved in transformation of lymphocytes but is dispensable for replication
Authors: Lupiani Blanca , Lee Lucy F. , Cui Xiaoping , Gimeno Isabel , Anderson Amy , Morgan Robin W. , Silva Robert F. , Witter Richard L. , Kung Hsing-Jien , Reddy Sanjay M.
Published: August 2004

Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in T cell lymphomas in visceral organs and peripheral nerves. Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV encode a basic leucine zipper protein, Meq, which structurally resembles the Jun/Fos family of transcriptional activators. Meq is consistently expressed in MDV-induced tumor cells and has been suggested as the MDV-associated oncogene. To study the function of Meq, we have generated an rMd5ΔMeq virus by deleting both copies of themeqgene from the genome of a very virulent strain of MDV. Growth curves in cultured fibroblasts indicated that Meq is dispensable forin vitrovirus replication.In vivoreplication in lymphoid organs and feather follicular epithelium was also not impaired, suggesting that Meq is dispensable for lytic infection in chickens. Reactivation of the rMd5ΔMeq virus from peripheral blood lymphocytes was reduced, suggesting that Meq is involved but not essential for latency. Pathogenesis experiments showed that the rMd5ΔMeq virus was fully attenuated in chickens because none of the infected chickens developed Marek's disease-associated lymphomas, suggesting that Meq is involved in lymphocyte transformation. A revertant virus that restored the expression of themeqgene, showed properties similar to those of the parental virus, confirming that Meq is involved in transformation but not in lytic replication in chickens.
Inhibitor design by wrapping packing defects in HIV-1 proteins
Authors: Fernández Ariel , Rogale Kristina , Scott Ridgway , Scheraga Harold A.
Published: August 2004

Two viral proteins, HIV-1 protease and HIV-1 integrase, have been targeted for inhibitor design to prevent assembly and maturation of HIV-1 virions. The enzymatic mechanism of these proteins involves side-chain groups that serve as general acids or bases. Furthermore, catalytic activity requires that water be removed from the microenvironment surrounding the chemical reaction site or be constrained to serve as an activated nucleophile. Here, we identify previously unrecognized structural features that promote water removal from polar catalytic regions. Packing defects in the form of hydrogen bonds that are insufficiently dehydrated intramolecularly, named “dehydrons,” are strategically placed in the structure to induce an anhydrous enzymatic pathway. Dehydrons become electrostatically enhanced and stabilized upon further desolvation. Thus, packing defects act synergistically with the polar active groups to enhance the enzymatic electrostatics. However, because dehydrons are sticky, they constitute targets for inhibitor design. We noticed that inhibitors attach to polar surfaces by further desolvating dehydrons, thus blocking the active sites or the sites involved in harnessing the substrate. The dehydrons are thus required for functional reasons, making them suitable targets. The differences in success when targeting HIV-1 protease, feline immunodeficiency virus protease, and HIV-1 integrase are rationalized in terms of the dehydron distribution, revealing possible improvements in the targeting strategy. Principles of design optimization are proposed to create an inhibitor that can be neutralized only at the expense of the loss of catalytic function. The possibility of using drugs that wrap dehydrons to block protein–protein associations is also discussed.
Recruitment of the ArgR/Mcm1p repressor is stimulated by the activator Gcn4p: A self-checking activation mechanism
Authors: Yoon Sungpil , Govind Chhabi K. , Qiu Hongfang , Kim Soon-ja , Dong Jinsheng , Hinnebusch Alan G.
Published: August 2004

Transcription of the arginine biosynthetic gene
ARG1
is repressed by the ArgR/Mcm1p complex in arginine-replete cells and activated by Gcn4p, a transcription factor induced by starvation for any amino acid. We show that all four subunits of the arginine repressor are recruited to
ARG1
by Gcn4p in cells replete with arginine but starved for isoleucine/valine. None of these proteins is recruited to the Gcn4p target genes
ARG4
and
SNZ1
, which are not regulated by ArgR/Mcm1p. Mcm1p and Arg80p were found in a soluble complex lacking Arg81p and Arg82p, and both Mcm1p and Arg80p were efficiently recruited to
ARG1
in wild-type cells in the presence or absence of exogenous arginine, and also in
arg81
Δ cells. By contrast, the recruitment of Arg81p and Arg82p was stimulated by exogenous arginine. These findings suggest that Gcn4p constitutively recruits an Mcm1p/Arg80p heterodimer and that efficient assembly of a functional repressor also containing Arg81p and Arg82p occurs only in arginine excess. By recruiting an arginine-regulated repressor, Gcn4p can precisely modulate its activation function at
ARG1
according to the availability of arginine.
Evolvability is a selectable trait
Authors: Earl David J. , Deem Michael W.
Published: August 2004

Concomitant with the evolution of biological diversity must have been the evolution of mechanisms that facilitate evolution, because of the essentially infinite complexity of protein sequence space. We describe how evolvability can be an object of Darwinian selection, emphasizing the collective nature of the process. We quantify our theory with computer simulations of protein evolution. These simulations demonstrate that rapid or dramatic environmental change leads to selection for greater evolvability. The selective pressure for large-scale genetic moves such as DNA exchange becomes increasingly strong as the environmental conditions become more uncertain. Our results demonstrate that evolvability is a selectable trait and allow for the explanation of a large body of experimental results.
How cholesterol homeostasis is regulated by plasma membrane cholesterol in excess of phospholipids
Authors: Lange Yvonne , Ye Jin , Steck Theodore L.
Published: August 2004

How do cells sense and control their cholesterol levels? Whereas most of the cell cholesterol is located in the plasma membrane, the effectors of its abundance are regulated by a small pool of cholesterol in the endoplasmic reticulum (ER). The size of the ER compartment responds rapidly and dramatically to small changes in plasma membrane cholesterol around the normal level. Consequently, increasing plasma membrane cholesterol
in vivo
from just below to just above the basal level evoked an acute (<2 h) and profound (≈20-fold) decrease in ER 3-hydroxy-3-methylglutaryl-CoA reductase activity
in vitro
. We tested the hypothesis that the sharply inflected ER response to cholesterol is governed by the thermodynamic activity (fugacity) of plasma membrane cholesterol. The following two independent measures of plasma membrane cholesterol activity in human red cells and fibroblasts were used: susceptibility to cholesterol oxidase and cholesterol transfer to cyclodextrin. Both indicators revealed a threshold at the physiologic set point of plasma membrane cholesterol. Incrementing the phospholipid compartment in the plasma membrane with lysophosphatidylcholine, previously shown to decrease cholesterol oxidase susceptibility, reduced the transfer of plasma membrane cholesterol to cyclodextrin and to the ER. Conversely, the membrane intercalator,
n
-octanol, increased cholesterol oxidation, transfer, and ER pool size, perhaps by displacing cholesterol from plasma membrane phospholipids. We conclude that the activity of the fraction of cholesterol in excess of other plasma membrane lipids sets the cholesterol level in the ER. Cholesterol-sensitive elements therein respond by nulling the active plasma membrane pool, thereby keeping the cholesterol matched to the other plasma membrane lipids.
The role of uncoupling protein 3 in fatty acid metabolism: protection against lipotoxicity?
Authors: Schrauwen Patrick
Published: August 2004

The physiological function of the mitochondrial uncoupling protein (UCP), UCP3, is still under debate. There is, however, ample evidence to indicate that, in contrast to UCP1, the primary function of UCP3 is not the dissipation of energy. Rather, several lines of evidence suggest that UCP3 is associated with cellular fatty acid metabolism. The highest levels of expression of UCP3 have been found in type 2 glycolytic muscle fibres, and fasting and high-fat diets up regulate UCP3. This up-regulation is most pronounced in muscle with a low fat oxidative capacity. Acute exercise also up regulates UCP3, and this effect has been shown to be a result of the exercise-induced increase in plasma fatty acid levels. In contrast, regular physical activity, which increases fat oxidative capacity, reduces UCP3 content. Based on these data it has been postulated that UCP3 functions to export those fatty acids that cannot be oxidized from the mitochondrial matrix, in order to prevent fatty acid accumulation inside the matrix. Several experiments have been conducted to test this hypothesis. Blocking carnitine palmitoyltransferase 1, thereby reducing fat oxidative capacity, rapidly induces UCP3. High-fat diets, which increase the mitochondrial supply of fatty acids, also up regulate UCP. However, feeding a similar amount of medium-chain fatty acids, which can be oxidized inside the mitochondrial matrix and therefore does not need to be exported from the matrix, does not affect UCP3 protein levels. In addition, UCP3 is increased in patients with defective β-oxidation and is reduced after restoring oxidative capacity. In conclusion, it is suggested that UCP3 has an important physiological function in facilitating outward transport from the mitochondrial matrix of fatty acid anions that cannot be oxidized, thereby protecting against lipid-induced mitochondrial damage.
Regulation of fatty acid transport by fatty acid translocase/CD36
Authors: Bonen Arend , Campbell Shannon E. , Benton Carley R. , Chabowski Adrian , Coort Susan L. M. , Han Xiao-Xia , Koonen Debby P. Y. , Glatz Jan F. C. , Luiken Joost J. F. P.
Published: August 2004

Fatty acid (FA) translocase (FAT)/CD36 is a key protein involved in regulating the uptake of FA across the plasma membrane in heart and skeletal muscle. A null mutation of FAT/CD36 reduces FA uptake rates and metabolism, while its overexpression increases FA uptake rates and metabolism. FA uptake into the myocyte may be regulated (a) by altering the expression of FAT/CD36, thereby increasing the plasmalemmal content of this protein (i.e. streptozotocin-induced diabetes, chronic muscle stimulation), or (b) by relocating this protein to the plasma membrane, without altering its expression (i.e. obese Zucker rats). By repressing FAT/CD36 expression, and thereby lowering the plasmalemmal FAT/CD36 (i.e. leptin-treated animals), the rate of FA transport is reduced. Within minutes of beginning muscle contraction or being exposed to insulin FA transport is increased. This increase is a result of the contraction- and insulin-induced translocation of FAT/CD36 from an intracellular depot to the cell surface. Neither PPARα nor PPARγ activation alter FAT/CD36 expression in muscle, despite the fact that PPARα activation increases FAT/CD36 by 80% in liver. A novel observation is that FAT/CD36 also appears to be involved in mitochondrial FA oxidation, as this protein is located on the mitochondrial membrane and seems to be required to participate in moving FA across the mitochondrial membrane. Clearly, FAT/CD36 has an important role in FA homeostasis in skeletal muscle and the heart.
Studies of plasma membrane fatty acid-binding protein and other lipid-binding proteins in human skeletal muscle
Authors: Roepstorff C. , Wulff Helge J. , Vistisen B. , Kiens B.
Published: August 2004

The first putative fatty acid transporter identified was plasma membrane fatty acid-binding protein (FABPpm). Later it was demonstrated that this protein is identical to the mitochondrial isoform of the enzyme aspartate aminotransferase. In recent years data from several cell types have emerged, indicating that FABPpmplays a role in the transport of long-chain saturated and unsaturated fatty acids. In the limited number of studies in human skeletal muscle it has been demonstrated that dietary composition and exercise training can influence the content of FABPpm. Ingestion of a fat-rich diet induces an increase in FABPpmprotein content in human skeletal muscle in contrast to the decrease seen during consumption of a carbohydrate-rich diet. A similar effect of a fat-rich diet is also observed for cytosolic fatty acid-binding protein and fatty acid translocase/CD36 protein expression. Exercise training up regulates FABPpmprotein content in skeletal muscle, but only in male subjects; no significant differences were observed in muscle FABPpmcontent in a cross-sectional study of female volunteers of varying training status, even though muscle FABPpmcontent did not depend on gender in the untrained state. A higher utilization of plasma long-chain fatty acids during exercise in males compared with females could explain the gender-dependent influence of exercise training on FABPpm. The mechanisms involved in the regulation of the function and expression of FABPpmprotein remain to be clarified.