bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensitivity of some Australian animals to sodium fluoroacetate (1080): additional species and populations, and some ecological considerations
Authors: Twigg Laurie E. , Martin Gary R. , Eastman Alan F. , King the late Dennis R. , Kirkpatrick Winifred E.
Published: December 2003

The sensitivity to fluoroacetate (1080) of a number of species of rodents and dasyurids with and without evolutionary exposure to fluoroacetate-bearing vegetation was determined. Rattus fuscipes, and species of Pseudomys from populations with exposure to this vegetation, were particularly tolerant to fluoroacetate. However, the level of tolerance varied among the different populations of each species, depending on the degree to which the toxic plants were present in their microhabitat. The tolerance of the F1 offspring of sensitive R. fuscipes (South Australia) crossed with tolerant conspecifics from Western Australia was mid-range between those of the parental populations. The sensitivity of introduced R. rattus and Mus domesticus from areas with fluoroacetate-producing plants in Western Australia was similar to that reported elsewhere for these rodents. This suggests that their relatively short coexistence with the toxic plants has had little obvious impact on their level of sensitivity to fluoroacetate. The dibbler, Parantechinus apicalis, which coexists with the toxic vegetation, was exceptionally tolerant for a native carnivore/insectivore (LD50 ~35 mg 1080 kg–1). In contrast, however, Phascogale tapoatafa from southern Western Australia was more sensitive to 1080 than was expected, with an estimated LD50 of 7 mg 1080 kg–1. Although the level of tolerance to fluoroacetate was seen to vary depending on the level of exposure of each species/population to fluoroacetate-bearing vegetation, our findings provide further evidence of the evolutionary impact that fluoroacetate-producing plants appear to have had on the genetic composition of indigenous Australian fauna.
Role of serotonin in cocaine effects in mice with reduced dopamine transporter function
Authors: Mateo Yolanda , Budygin Evgeny A. , John Carrie E. , Jones Sara R.
Published: January 2004

The mesolimbic dopaminergic system, especially the nucleus accumbens, has received attention for its involvement in the reinforcing and addictive properties of cocaine and other drugs of abuse. It is generally accepted that the ability of cocaine to inhibit the dopamine transporter (DAT) is directly related to its reinforcing actions. However, mice with a genetic deletion of the DAT (DAT-KO mice) still experience the rewarding effects of cocaine. These behavioral findings suggest that there is an alternate site for cocaine reinforcement. We demonstrate here that modulation of the serotonergic system in the ventral tegmental area, where the mesolimbic dopamine system originates, is a target of cocaine action. The ultimate effect of this serotonin mechanism in animal models with sustained elevations of dopamine may be a feed-forward enhancement of dopamine levels in the nucleus accumbens.
Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects
Authors: Schäcke Heike , Schottelius Arndt , Döcke Wolf-Dietrich , Strehlke Peter , Jaroch Stefan , Schmees Norbert , Rehwinkel Hartmut , Hennekes Hartwig , Asadullah Khusru
Published: January 2004

Glucocorticoids (GCs) are the most commonly used antiinflammatory and immunosuppressive drugs. Their outstanding therapeutic effects, however, are often accompanied by severe and sometimes irreversible side effects. For this reason, one goal of research in the GC field is the development of new drugs, which show a reduced side-effect profile while maintaining the antiinflammatory and immunosuppressive properties of classical GCs. GCs affect gene expression by both transactivation and transrepression mechanisms. The antiinflammatory effects are mediated to a major extent via transrepression, while many side effects are due to transactivation. Our aim has been to identify ligands of the GC receptor (GR), which preferentially induce transrepression with little or no transactivating activity. Here we describe a nonsteroidal selective GR-agonist, ZK 216348, which shows a significant dissociation between transrepression and transactivation both
in vitro
and
in vivo
. In a murine model of skin inflammation, ZK 216348 showed antiinflammatory activity comparable to prednisolone for both systemic and topical application. A markedly superior side-effect profile was found with regard to increases in blood glucose, spleen involution, and, to a lesser extent, skin atrophy; however, adrenocorticotropic hormone suppression was similar for both compounds. Based on these findings, ZK 216348 should have a lower risk, e.g., for induction of diabetes mellitus. The selective GR agonists therefore represent a promising previously undescribed class of drug candidates with an improved therapeutic index compared to classical GCs. Moreover, they are useful tool compounds for further investigating the mechanisms of GR-mediated effects.
Distinct forms of cholinergic modulation in parallel thalamic sensory pathways
Authors: Mooney D. M. , Zhang L. , Basile C. , Senatorov V. V. , Ngsee J. , Omar A. , Hu B.
Published: January 2004

Mammalian thalamus is a critical site where early perception of sensorimotor signals is dynamically regulated by acetylcholine in a behavioral state-dependent manner. In this study, we examined how synaptic transmission is modulated by acetylcholine in auditory thalamus where sensory relay neurons form parallel lemniscal and nonlemniscal pathways. The former mediates tonotopic relay of acoustic signals, whereas the latter is involved in detecting and transmitting auditory cues of behavioral relevance. We report here that activation of cholinergic muscarinic receptors had opposite membrane effects on these parallel synaptic pathways. In lemniscal neurons, muscarine induced a sustained membrane depolarization and tonic firing by closing a linear K
+
conductance. In contrast, in nonlemniscal neurons, muscarine evoked a membrane hyperpolarization by opening a voltage-independent K
+
conductance. Depending on the level of membrane hyperpolarization and the strength of local synaptic input, nonlemniscal neurons were either suppressed or selectively engaged in detecting and transmitting synchronized synaptic input by firing a high-frequency spike burst. Immunohistochemical and Western blotting experiments showed that nonlemniscal neurons predominantly expressed M2 muscarinic receptors, whereas lemniscal cells had a significantly higher level of M1 receptors. Our data indicate that cholinergic modulation in the thalamus is pathway-specific. Enhanced cholinergic tone during behavioral arousal or attention may render synaptic transmission in nonlemniscal thalamus highly sensitive to the context of local synaptic activities.
Structure of HrcQ
B
-C, a conserved component of the bacterial type III secretion systems
Authors: Fadouloglou Vasiliki E. , Tampakaki Anastasia P. , Glykos Nicholas M. , Bastaki Marina N. , Hadden Jonathan M. , Phillips Simon E. , Panopoulos Nicholas J. , Kokkinidis Michael
Published: January 2004

Type III secretion systems enable plant and animal bacterial pathogens to deliver virulence proteins into the cytosol of eukaryotic host cells, causing a broad spectrum of diseases including bacteremia, septicemia, typhoid fever, and bubonic plague in mammals, and localized lesions, systemic wilting, and blights in plants. In addition, type III secretion systems are also required for biogenesis of the bacterial flagellum. The HrcQ
B
protein, a component of the secretion apparatus of
Pseudomonas syringae
with homologues in all type III systems, has a variable N-terminal and a conserved C-terminal domain (HrcQ
B
-C). Here, we report the crystal structure of HrcQ
B
-C and show that this domain retains the ability of the full-length protein to interact with other type III components. A 3D analysis of sequence conservation patterns reveals two clusters of residues potentially involved in protein–protein interactions. Based on the analogies between HrcQ
B
and its flagellum homologues, we propose that HrcQ
B
-C participates in the formation of a C-ring-like assembly.
U4 small nuclear RNA can function in both the major and minor spliceosomes
Authors: Shukla Girish C. , Padgett Richard A.
Published: January 2004

U4 small nuclear RNA (snRNA) and U6 snRNA form a base-paired di-snRNP complex that is essential for pre-mRNA splicing of the major class of metazoan nuclear introns. The functionally analogous but highly diverged U4atac and U6atac snRNAs form a similar complex that is involved in splicing of the minor class of introns. Previous results with mutants of U6atac in which a substructure was replaced by the analogous structure from U6 snRNA suggested that wild-type U4 snRNA might be able to interact productively with the mutant U6atac snRNA. Here we show that a mutant U4 snRNA designed to base pair with a mutant U6atac snRNA can activate U12-dependent splicing when coexpressed in an
in vivo
genetic suppression assay. This genetic interaction could also be demonstrated in an
in vitro
crosslinking assay. These results show that a U4/U6atac di-snRNP can correctly splice a U12-dependent intron and suggest that the specificity for spliceosome type resides in the U6 and U6atac small nuclear ribonucleoproteins. Further experiments suggest that expression of a mutant U4 snRNA that can bind to wild-type U6atac snRNA alters the specificity of some splice sites, providing an evolutionary rationale for maintaining two U4-like snRNAs.
Crystal structure of a central stalk subunit C and reversible association/dissociation of vacuole-type ATPase
Authors: Iwata Momi , Imamura Hiromi , Stambouli Elizabeth , Ikeda Chiyo , Tamakoshi Masatada , Nagata Koji , Makyio Hisayoshi , Hankamer Ben , Barber Jim , Yoshida Masasuke , Yokoyama Ken , Iwata So
Published: January 2004

The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of
Thermus thermophilus
V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-Å resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments. The result shows that V-ATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the V
1
(soluble) and the V
o
(membrane bound) domains. Subunit C, attached to the V
o
domain, seems to have a socket like function in attaching the central-stalk subunits of the V
1
domain. This architecture seems essential for the reversible association/dissociation of the V
1
and the V
o
domains, unique for V-ATPase activity regulation.
A human complement receptor 1 polymorphism that reducesPlasmodium falciparumrosetting confers protection against severe malaria
Authors: Cockburn Ian A. , Mackinnon Margaret J. , O'Donnell Angela , Allen Stephen J. , Moulds Joann M. , Baisor Moses , Bockarie Moses , Reeder John C. , Rowe J. Alexandra
Published: January 2004

Parasitized red blood cells (RBCs) from children suffering from severe malaria often adhere to complement receptor 1 (CR1) on uninfected RBCs to form clumps of cells known as “rosettes.” Despite a well documented association between rosetting and severe malaria, it is controversial whether rosetting is a cause or a correlate of parasite virulence. CR1-deficient RBC show greatly reduced rosetting; therefore, we hypothesized that, if rosetting is a direct cause of malaria pathology, CR1-deficient individuals should be protected against severe disease. In this study, we show that RBC CR1 deficiency occurs in up to 80% of healthy individuals from the malaria-endemic regions of Papua New Guinea. This RBC CR1 deficiency is associated with polymorphisms in the CR1 gene and, unexpectedly, with α-thalassemia, a common genetic disorder in Melanesian populations. Analysis of a case-control study demonstrated that the CR1 polymorphisms and α-thalassemia independently confer protection against severe malaria. We have therefore identified CR1 as a new malaria resistance gene and provided compelling evidence that rosetting is an important parasite virulence phenotype that should be a target for drug and vaccine development.
A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms
Authors: Portevin Damien , de Sousa-D'Auria Célia , Houssin Christine , Grimaldi Christine , Chami Mohamed , Daffé Mamadou , Guilhot Christophe
Published: January 2004

Mycolic acids are major and specific constituents of the cell envelope of
Corynebacterineae
, a suborder of bacterial species including several important human pathogens such as
Mycobacterium tuberculosis, Mycobacterium leprae
, or
Corynebacterium diphtheriae
. These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria. The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades. By
in silico
analysis of various mycobacterial genomes, we identified a candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction. Orthologs of this enzyme were found in other
Corynebacterineae
species. A
Corynebacterium glutamicum
strain with a deletion in the
pks13
gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors. This mutant strain displayed an altered cell envelope structure. We showed that the
pks13
gene was essential for the survival of
Mycobacterium smegmatis
. A conditional
M. smegmatis
mutant carrying its only copy of
pks13
on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature. These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against
Corynebacterineae
.
Could CO
2
-induced land-cover feedbacks alter near-shore upwelling regimes?
Authors: Diffenbaugh Noah S. , Snyder Mark A. , Sloan Lisa C.
Published: January 2004

The response of marine and terrestrial environments to global changes in atmospheric carbon dioxide (CO
2
) concentrations will likely be governed by both responses to direct environmental forcing and responses to Earth-system feedbacks induced by that forcing. It has been proposed that anthropogenic greenhouse forcing will intensify coastal upwelling in eastern boundary current regions [Bakun, A. (1990)
Science
247, 198–201]. Focusing on the California Current, we show that biophysical land-cover–atmosphere feedbacks induced by CO
2
radiative forcing enhance the radiative effects of CO
2
on land–sea thermal contrast, resulting in changes in eastern boundary current total seasonal upwelling and upwelling seasonality. Specifically, relative to CO
2
radiative forcing, land-cover–atmosphere feedbacks lead to a stronger increase in peak- and late-season near-shore upwelling in the northern limb of the California Current and a stronger decrease in peak- and late-season near-shore upwelling in the southern limb. Such changes will impact both marine and terrestrial communities [Bakun, A. (1990)
Science
247, 198–201; Soto, C. G. (2001)
Rev. Fish Biol. Fish.
11, 181–195; and Agostini, V. N. & Bakun, A. (2002)
Fish. Oceanogr.
11, 129–142], and these and other Earth-system feedbacks should be expected to play a substantial role in shaping the response of eastern boundary current regions to CO
2
radiative forcing.
Hybridoma-free generation of monoclonal antibodies
Authors: Pasqualini Renata , Arap Wadih
Published: January 2004

Production of monoclonal antibodies requires immortalization of splenocytes by somatic fusion to a myeloma cell line partner (hybridomas). Although hybridomas can be immortal, they may depend on a feeder cell layer and may be genetically unstable. Since the inception of hybridoma technology, efforts to improve efficiency and stability of monoclonal antibody-producing cell lines have not brought about substantial progress. Moreover, suitable human multiple myeloma-derived cell lines for the production of human antibodies have been very difficult to develop. Here we report a strategy that greatly simplifies the generation of antibodies and eliminates the need for hybridomas. We show that splenocytes derived from transgenic mice harboring a mutant temperature-sensitive simian virus 40 large tumor antigen under the control of a mouse major histocompatibility promoter are conditionally immortal at permissive temperatures and produce monoclonal antibodies. This simple approach may become a method of choice for generation and production of both polyclonal and monoclonal antibodies with advantages in high-throughput discovery and antibody-based immunotherapy.
Mutant human cells with constitutive activation of NF-κB
Authors: Sathe Swati S. , Sizemore Nywana , Li Xiaoxia , Vithalani Kalpa , Commane Mairead , Swiatkowski Shannon M. , Stark George R.
Published: January 2004

We have used a genetic approach to generate eight different mutant human cell lines in which NF-κB is constitutively activated. These independent clones have different phenotypes and belong to several different genetic complementation groups. In one clone inhibitor of κB(IκB) kinase is constitutively active, but in the seven others it is not, despite the fact that IκB is degraded in all eight clones. Thus, IκB kinase-independent mechanisms of IκB degradation and NF-κB activation are predominant in these mutants. Biochemical analyses of the mutants revealed that they fall into at least five different categories, differing in the sets of upstream kinases that are activated, confirming multiple mechanisms of NF-κB activation. By introducing a retroviral cDNA library into the Ras C6 cell line, with constitutively active NF-κB, followed by selection for functional complementation, we isolated a cDNA encoding a C-terminal fragment of enolase 1 and identified it as negative regulator of NF-κB.
Variable ecological effects of hurricanes: The importance of seasonal timing for survival of lizards on Bahamian islands
Authors: Schoener Thomas W. , Spiller David A. , Losos Jonathan B.
Published: January 2004

Two recent hurricanes passed directly over the northern Bahamas 2 years apart, allowing a comparison of their effects on lizard populations inhabiting exactly the same islands. The hurricanes differed in two ways: one struck during the reproductive season and was relatively severe; the other struck after most reproduction had taken place and was milder. The late-season hurricane produced a significant relation between population reduction and lowness of the island that lasted at least through two seasons; the earlier hurricane produced no such relationship. The late-season hurricane wiped out populations of lizards on two islands (two of the three lowest) that the earlier hurricane failed to exterminate even though it was stronger. We relate these effects to the fact that the study lizards regenerated from the earlier hurricane only via the egg stage, whereas eggs were unavailable when the later storm struck and regeneration was via hatched lizards. We discriminate and illustrate four kinds of hurricanes, cross-classified by two contrasts: earlier vs. later and stronger vs. weaker. A later, stronger hurricane completely exterminated lizard populations at a second Bahamian site, whereas an earlier, weaker hurricane had no detectable effect at a third Bahamian site. We suggest that, in addition to severity, the timing of a hurricane as it coincides with reproductive scheduling or other phenological aspects may determine the magnitude of its effect on a variety of organisms.
Enhanced hippocampal noradrenaline and serotonin release in galanin-overexpressing mice after repeated forced swimming test
Authors: Yoshitake Takashi , Wang Fu-Hua , Kuteeva Eugenia , Holmberg Kristina , Yamaguchi Masatoshi , Crawley Jacqueline N. , Steiner Robert , Bartfai Tamas , Ögren Sven Ove , Hökfelt Tomas , Kehr Jan
Published: January 2004

Basal and forced swimming (FS) stress-induced release of noradrenaline (NA) and serotonin (5-HT) were determined by
in vivo
microdialysis in the ventral hippocampus of mice overexpressing galanin under the platelet-derived growth factor B promoter (GalOE/P) or under the dopamine β-hydroxylase promoter (GalOE/D) (only NA). WT mice served as controls. Intraventricular infusion of galanin significantly reduced basal extracellular NA in WT mice and in GalOE/P mice (albeit less so). Microdialysis sampling during a 10-min FS showed that NA and 5-HT release were elevated to 213% and 156%, respectively, in the GalOE/P group, whereas in the WT group the increases were only 127% and 119%, respectively. The second (repeated) 10-min FS (RFS) caused a marked enhancement of NA and 5-HT release in the GalOE/P mice to 344% and 275%, respectively. However, the RFS caused only a 192% increase of extracellular NA levels in the GalOE/D mice. Pretreatment with the putative peptidergic galanin receptor antagonist M35 almost completely blocked the elevation of NA and 5-HT levels in the GalOE/P after RFS. These results suggest that the NA and 5-HT hippocampal afferents in GalOE/P mice are hypersensitive to both conditioned and unconditioned stressful stimuli, such as FS, and that this effect is mediated by galanin receptors. The present findings support a role of galanin in the regulation of release of NA and 5-HT, two neurotransmitters involved in mood control.
Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells
Authors: Uddin Shahab , Ah-Kang Jeong , Ulaszek Jodie , Mahmud Dolores , Wickrema Amittha
Published: January 2004

p38α, p38β, p38γ, and p38δ are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38α and p38γ transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38δ mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38β is minimally expressed in early CD34+hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38α, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38α, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38α does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38α and p38δ isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor α, an inflammatory cytokine that is modulated by p38α, is expressed by differentiating erythroblasts and inhibition of p38α or tumor necrosis factor α results in reduction in differentiation. Taken together, our data demonstrate that both p38α and δ isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.
The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide ofStreptococcus pneumoniaein the marginal zone of mouse spleen
Authors: Kang Young-Sun , Kim Jae Y. , Bruening Sandra A. , Pack Maggi , Charalambous Anna , Pritsker Alla , Moran Thomas M. , Loeffler Jutta M. , Steinman Ralph M. , Park Chae Gyu
Published: January 2004

SIGN-R1, a recently discovered C-type lectin expressed at high levels on macrophages within the marginal zone of the spleen, mediates the uptake of dextran polysaccharides by these phagocytes. We now find that encapsulatedStreptococcus pneumoniaeare rapidly cleared by these macrophages from the bloodstream, and that capture also takes place when different cell lines express SIGN-R1 after transfection. To assess the role of the capsular polysaccharide ofS. pneumoniae(CPS) in the interaction of SIGN-R1 with pneumococci, we first studied binding and uptake of serotype 14 CPS in transfected cells. Binding was observed and was of a much higher avidity (3,000-fold) for CPS 14 than dextran. The CPSs from four different serotypes were also cleared by marginal zone macrophagesin vivo. To establish a role for SIGN-R1 in this uptake, we selectively down-regulated expression of the lectin by pretreatment of the mice with SIGN-R1 antibodies, including a newly generated hamster monoclonal called 22D1. For several days after this transient knockout, the marginal zone macrophages were unable to take up either CPSs or dextrans. Therefore, marginal zone macrophages in mice have a receptor that interacts with capsular pneumococcal polysaccharides, setting the stage for further studies of the functional consequences of this interaction.
Detection of bacteria in suspension by using a superconducting quantum interference device
Authors: Grossman H. L. , Myers W. R. , Vreeland V. J. , Bruehl R. , Alper M. D. , Bertozzi C. R. , Clarke John
Published: January 2004

We demonstrate a technique for detecting magnetically labeledListeria monocytogenesand for measuring the binding rate between antibody-linked magnetic particles and bacteria. This sensitive assay quantifies specific bacteria in a sample without the need to immobilize them or wash away unbound magnetic particles. In the measurement, we add 50-nm-diameter superparamagnetic magnetite particles, coated with antibodies, to an aqueous sample containingL. monocytogenes. We apply a pulsed magnetic field to align the magnetic dipole moments and use a high-transition temperature superconducting quantum interference device, an extremely sensitive detector of magnetic flux, to measure the magnetic relaxation signal when the field is turned off. Unbound particles randomize direction by Brownian rotation too quickly to be detected. In contrast, particles bound toL. monocytogenesare effectively immobilized and relax in about 1 s by rotation of the internal dipole moment. This Néel relaxation process is detected by the superconducting quantum interference device. The measurements indicate a detection limit of (5.6 ± 1.1) × 106L. monocytogenesin our sample volume of 20 μl. If the sample volume were reduced to 1 nl, we estimate that the detection limit could be improved to 230 ± 40L. monocytogenescells. Time-resolved measurements yield the binding rate between the particles and bacteria.
A site-specific factor interacts directly with its cognate RNA editing site in chloroplast transcripts
Authors: Miyamoto Tetsuya , Obokata Junichi , Sugiura Masahiro
Published: January 2004

RNA editing involves a variety of genetic systems and occurs by different mechanisms. In higher plant chloroplasts, specific sites of some transcripts are subject to C-to-U conversion. We have previously shown that site-specific trans-acting factors for
psbE
and
petB
mRNA editing bind corresponding cis-elements, which are located 5 nucleotides upstream from the editing site. Here we report that, by using mRNAs labeled either at the center of the upstream cis-element or at the editing site, the site-specific factors can be cross-linked with nucleotides at both positions. Mutations of nucleotides in the proximal region of the editing site revealed a correlation between editing activity and cross-linking efficiency of factors with the editing site, even though cross-linking with the upstream cis-element was unaffected. These observations suggest that the site-specific factor binds stably to the upstream ciselement, whereas it interacts weakly with the editing site. This finding raises the intriguing possibility that the site-specific factor is involved in both site-determination and C-to-U conversion in chloroplast RNA editing.
A functional genomic screen for cardiogenic genes using RNA interference in developing
Drosophila
embryos
Authors: Kim Yong-Ou , Park Sang-Joon , Balaban Robert S. , Nirenberg Marshall , Kim Yongsok
Published: January 2004

Identifying genetic components is an essential step toward understanding complex developmental processes. The primitive heart of the fruit fly, the dorsal vessel, which is a hemolymph-pumping organ, has provided a unique model system to identify cardiogenic genes and to further our understanding of the molecular mechanisms of cardiogenesis. Using RNA interference in developing
Drosophila
embryos, we performed a genomewide search for cardiogenic genes. Through analyses of the >5,800 genes that cover ≈40% of all predicted
Drosophila
genes, we identified a variety of genes encoding transcription factors and cell signaling proteins required for different steps during heart development. Analysis of mutant heart phenotypes and identified genes suggests that the
Drosophila
heart tube is segmentally patterned, like axial patterning, but assembled with regional modules. One of the identified genes,
simjang
, was further characterized. In the
simjang
mutant embryo, we found that within each segment a subset of cardial cells is missing. Interestingly, the
simjang
gene encodes a protein that is a component of the chromatin remodeling complex recruited by methyl-CpG-DNA binding proteins, suggesting that epigenetic information is crucial for specifying cardiac precursors. Together, these studies not only identify key regulators but also reveal mechanisms underlying heart development.
Selective abolition of pancreatic RNase binding to its inhibitor protein
Authors: Kumar Kapil , Brady Michael , Shapiro Robert
Published: January 2004

We have modified RNase inhibitor (RI) protein so that it no longer detectably binds pancreatic RNases but retains near-native affinity for human angiogenin (ANG). TheKivalue for RNase A is increased by a factor of >108, from 36 fM to >4 μM, and the selectivity factor for ANG is now >109. This dramatic change was achieved by remodeling the human RI loop segment Cys-408 -Leu-409 -Gly-410, which makes minor interactions with pancreatic RNase but does not contact ANG. The modifications selected were designed to sterically hinder docking of the undesired ligand. Three of the variants tested (C408W, G410W, and C408W/G410W) bind RNase A with almost the same avidity as WT RI. However, combination of the 408/410 double Trp replacement with deletion of the intervening residue, Leu-409, was sufficient to abolish inhibition of RNase A and human pancreatic RNase. TheKivalue for ANG with the deletion variant is 1.1 fM, only 2-fold higher than with WT RI. This variant may have potential utility both as an anticancer drug targeting ANG and as a tool for the investigation of the biological function of ANG. More generally, these findings demonstrate that a protein-protein interaction can be effectively and specifically disrupted by redesigning an interface region that makes no major energetic contribution to complex stability. This finding, in turn, may have implications for the development of small molecules that modulate protein-protein interactions.
Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3)
Authors: Long Jianyin , Wang Guannan , Matsuura Isao , He Dongming , Liu Fang
Published: January 2004

Smad proteins play pivotal roles in mediating the transforming growth factor β (TGF-β) transcriptional responses. We show in this report that PIAS3, a member of the protein inhibitor of activated STAT (PIAS) family, activates TGF-β/Smad transcriptional responses. PIAS3 interacts with Smad proteins, most strongly with Smad3. PIAS3 and Smad3 interact with each other at the endogenous protein level in mammalian cells and also
in vitro
, and the association occurs through the C-terminal domain of Smad3. We further show that PIAS3 can interact with the general coactivators p300/CBP, the first evidence that a PIAS protein can associate with p300/CBP. In contrast, PIASy, which inhibits Smad transcriptional activity and other transcriptional responses, is unable to interact with p300/CBP. The RING domain of PIAS3 is essential for interaction with p300/CBP, and a RING domain mutant PIAS3, which cannot bind p300/CBP, no longer activates TGF-β/Smad-dependent transcription. Furthermore, we show that PIAS3, Smad3, and p300 can form a ternary complex, which is markedly increased by TGF-β treatment. Taken together, our studies indicate that on TGF-β treatment, PIAS3 can form a complex with Smads and p300/CBP and activate Smad transcriptional activity.
The crystal structure of
Pseudomonas
avirulence protein AvrPphB: A papain-like fold with a distinct substrate-binding site
Authors: Zhu Minfeng , Shao Feng , Innes Roger W. , Dixon Jack E. , Xu Zhaohui
Published: January 2004

AvrPphB is an avirulence (Avr) protein from the plant pathogen
Pseudomonas syringae
that can trigger a disease-resistance response in a number of host plants including
Arabidopsis
. AvrPphB belongs to a novel family of cysteine proteases with the charter member of this family being the
Yersinia
effector protein YopT. AvrPphB has a very stringent substrate specificity, catalyzing a single proteolytic cleavage in the
Arabidopsis
serine/threonine kinase PBS1. We have determined the crystal structure of AvrPphB by x-ray crystallography at 1.35-Å resolution. The structure is composed of a central antiparallel β-sheet, with α-helices packing on both sides of the sheet to form a two-lobe structure. The core of this structure resembles the papain-like cysteine proteases. The similarity includes the AvrPphB active site catalytic triad of Cys-98, His-212, and Asp-227 and the oxyanion hole residue Asn-93. Based on analogy with inhibitor complexes of the papain-like proteases, we propose a model for the substrate-binding mechanism of AvrPphB. A deep and positively charged pocket (S2) and a neighboring shallow surface (S3) likely bind to aspartic acid and glycine residues in the substrate located two (P2) and three (P3) residues N terminal to the cleavage site, respectively. Further implications about the specificity of plant pathogen recognition are also discussed.
An approach to genomewide screens of expressed small interfering RNAs in mammalian cells
Authors: Zheng Lianxing , Liu Jun , Batalov Sergei , Zhou Demin , Orth Anthony , Ding Sheng , Schultz Peter G.
Published: January 2004

To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a gene-specific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA duplex with a uridine overhang on each 3′ terminus. A single-step PCR protocol has been developed by using this dual promoter system that allows the production of siRNA expression cassettes in a high-throughput manner. We have shown that siRNAs transcribed by either the dual promoter vector or siRNA expression cassettes can induce strong and gene-specific suppression of both endogenous genes and ectopically expressed genes in mammalian cells. Furthermore, we have constructed an arrayed siRNA expression cassette library that targets >8,000 genes with two siRNA sequences per gene. A high-throughput screen of this library has revealed both known and unique genes involved in the NF-κB signaling pathway.
Thyroid hormone controls the development of connections between the spinal cord and limbs during
Xenopus laevis
metamorphosis
Authors: Marsh-Armstrong Nicholas , Cai Liquan , Brown Donald D.
Published: January 2004

During premetamorphic stages,
Xenopus laevis
tadpoles expressing either a dominant-negative thyroid hormone (TH) receptor or a type-III iodothyronine deiodinase transgene in the nervous system have reduced TH-induced proliferation in the spinal cord and produce fewer hindlimb-innervating motorneurons. During prometamorphic stages, innervation of the hindlimbs is reduced, and few functional neuromuscular connections are formed. By metamorphic climax, limb movement is impaired, ranging from uncoordinated leg swimming to complete quadriplegia. This phenotype is due to transgene action in the tadpole spinal cord. The requirement of TH for neurogenesis during premetamorphosis is the earliest TH-regulated process reported to date in the sequence of metamorphic changes in anurans. The muscle formed during limb growth was previously shown to be a direct target of TH control. Here, we show that the same is true of the development of spinal cord cells that innervate the limbs.
Electroporation and RNA interference in the rodent retina
in vivo
and
in vitro
Authors: Matsuda Takahiko , Cepko Constance L.
Published: January 2004

The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain- and loss-of-function studies
in vivo
and
in vitro
in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina
in vivo
, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants
in vitro
by high-voltage pulses.
Epstein–Barr virus latent infection membrane protein 1 TRAF-binding site induces NIK/IKKα-dependent noncanonical NF-κB activation
Authors: Luftig Micah , Yasui Teruhito , Soni Vishal , Kang Myung-Soo , Jacobson Nils , Cahir-McFarland Ellen , Seed Brian , Kieff Elliott
Published: January 2004

Epstein–Barr virus (EBV) latent infection membrane protein 1 (LMP1)-induced NF-κB activation is important for infected cell survival. LMP1 activates NF-κB, in part, by engaging tumor necrosis factor (TNF) receptor-associated factors (TRAFs), which also mediate NF-κB activation from LTβR and CD40. LTβR and CD40 activation of p100/NF-κB2 is now known to be NIK/IKKα-dependent and IKKβ/IKKγ independent. In the experiments described here, we found that EBV LMP1 induced p100/NF-κB2 processing in human lymphoblasts and HEK293 cells. LMP1-induced p100 processing was NIK/IKKα dependent and IKKβ/IKKγ independent. Furthermore, the LMP1 TRAF-binding site was required for p100 processing and p52 nuclear localization, whereas the LMP1 death domain-binding site was not. Moreover, the LMP1 TRAF-binding site preferentially caused RelB nuclear accumulation. In murine embryo fibroblasts (MEFs), IKKβ was essential for LMP1 up-regulation of macrophage inflammatory protein (MIP)-2, TNFα, I-TAC, ELC, MIG, and CXCR4 RNAs. Interestingly, in IKKα knockout MEFs, LMP1 hyperinduced MIP-2, TNFα, and I-TAC expression, consistent with a role for IKKα in down-modulating canonical IKKβ activation or its effects. In contrast, LMP1 failed to up-regulate CXCR4 and MIG RNA in IKKα knockout MEFs, indicating a dependence on noncanonical IKKα activation. Furthermore, LMP1 up-regulation of MIP-2 RNA in MEFs was both IKKβ- and IKKγ-dependent, whereas LMP1 upregulation of MIG and I-TAC RNA was fully IKKγ independent. Thus, LMP1 induces typical canonical IKKβ/IKKγ-dependent, atypical canonical IKKβ-dependent/IKKγ-independent, and noncanonical NIK/IKKα-dependent NF-κB activations; NIK/IKKα-dependent NF-κB activation is principally mediated by the LMP1 TRAF-binding site.
Latent infection membrane protein transmembrane FWLY is critical for intermolecular interaction, raft localization, and signaling
Authors: Yasui Teruhito , Luftig Micah , Soni Vishal , Kieff Elliott
Published: January 2004

Relatively little is known about the biochemical mechanisms through which the Epstein–Barr virus latent infection integral membrane protein 1 (LMP1) transmembrane domains cause constitutive LMP1 aggregation and continuous cytoplasmic C terminus-mediated signal transduction. We now evaluate the role of the three consecutive LMP1 hydrophobic transmembrane pairs, transmembrane domains (TM)1-2, TM3-4, and TM5-6, in intermolecular aggregation and NF-κB activation. LMP1TM1-2 enabled ≈40% of wild-type LMP1 cytoplasmic domain-mediated NF-κB activation, whereas TM3-4 or TM5-6 assayed in parallel had almost no effect independent of LMP1TM1-2. Alanine mutagenesis of conserved residues in LMP1TM1-2 identified FWLY38–41to be critical for LMP1TM1-2 intermolecular association with LMP1TM3-6. Further, in contrast to wild-type LMP1, LMP1 with FWLY38–41mutated to AALA38–41did not (i) significantly partition to lipid Rafts or Barges and effectively intermolecularly associate, (ii) enable cytoplasmic C terminus engagement of tumor necrosis factor receptor-associated factor 3, (iii) activate NF-κB, and thereby (iv) induce tumor necrosis factor receptor-associated factor 1 expression. Other LMP1 intermolecular associations were observed that involved LMP1TM1-2/LMP1TM1-2 or LMP1TM3-4/LMP1TM3-6 interactions; these probably also contribute to LMP1 aggregation. Because FWLY38–41was essential for LMP1-mediated signal transduction, and LMP1 activation of NF-κB is essential for proliferating B lymphocyte survival, inhibition of LMP1FWLY41-mediated LMP1/LMP1 intermolecular interactions is an attractive therapeutic target.
Genetic introgression as a potential to widen a species' niche: Insights from alpine
Carex curvula
Authors: Choler P. , Erschbamer B. , Tribsch A. , Gielly L. , Taberlet P.
Published: January 2004

Understanding what causes the decreasing abundance of species at the margins of their distributions along environmental gradients has drawn considerable interest, especially because of the recent need to predict shifts in species distribution patterns in response to climatic changes. Here, we address the ecological range limit problem by focusing on the sedge,
Carex curvula
, a dominant plant of high-elevation grasslands in Europe, for which two ecologically differentiated but crosscompatible taxa have been described in the Alps. Our study heuristically combines an extensive phytoecological survey of alpine plant communities to set the niche attributes of each taxon and a population genetic study to assess the multilocus genotypes of 177 individuals sampled in typical and marginal habitats. We found that ecological variation strongly correlates with genetic differentiation. Our data strongly suggest that ecologically marginal populations of each taxon are mainly composed of individuals with genotypes resulting from introgressive hybridization. Conversely, no hybrids were found in typical habitats, even though the two taxa were close enough to crossbreed. Thus, our results indicate that genotype integrity is maintained in optimal habitats, whereas introgressed individuals are favored in marginal habitats. We conclude that gene flow between closely related taxa might be an important, although underestimated, mechanism shaping species distribution along gradients.
The
Arabidopsis LUT1
locus encodes a member of the cytochrome P450 family that is required for carotenoid ε-ring hydroxylation activity
Authors: Tian Li , Musetti Valeria , Kim Joonyul , Magallanes-Lundback Maria , DellaPenna Dean
Published: January 2004

Lutein, a dihydroxy xanthophyll, is the most abundant carotenoid in plant photosynthetic tissues and plays crucial structural and functional roles in the light-harvesting complexes. Carotenoid β-and ε-hydroxylases catalyze the formation of lutein from α-carotene (β,ε-carotene). In contrast to the well studied β-hydroxylases that have been cloned and characterized from many organisms, the ε-hydroxylase has only been genetically defined by the
lut1
mutation in
Arabidopsis
. We have isolated the
LUT1
gene by positional cloning and found that, in contrast to all known carotenoid hydroxylases, which are the nonheme diiron monooxygenases,
LUT1
encodes a cytochrome P450-type monooxygenase, CYP97C1. Introduction of a null mutant allele of
LUT1, lut1
-
3
, into the β-hydroxylase 1/β-hydroxylase 2 (
b1 b2
) double-mutant background, in which both
Arabidopsis
β-hydroxylases are disrupted, yielded a genotype (
lut1
-
3 b1 b2
) in which all three known carotenoid hydroxylase activities are eliminated. Surprisingly, hydroxylated β-rings were still produced in
lut1
-
3 b1 b2
, suggesting that a fourth unknown carotenoid β-hydroxylase exists
in vivo
that is structurally unrelated to β-hydroxylase 1 or 2. A second chloroplast-targeted member of the CYP97 family, CYP97A3, is 49% identical to LUT1 and hypothesized as a likely candidate for this additional β-ring hydroxylation activity. Overall, LUT1 defines a class of carotenoid hydroxylases that has evolved independently from and uses a different mechanism than nonheme diiron β-hydroxylases.
Magnetism in dense hexagonal iron
Authors: Steinle-Neumann Gerd , Stixrude Lars , Cohen Ronald E.
Published: January 2004

The magnetic state of hexagonal close-packed iron has been the subject of debate for more than three decades. Although Mössbauer measurements find no evidence of the hyperfine splitting that can signal the presence of magnetic moments, density functional theory predicts an antiferromagnetic (afm) ground state. This discrepancy between theory and experiment is now particularly important because of recent experimental findings of anomalous splitting in the Raman spectra and the presence of superconductivity in hexagonal close-packed iron, which may be caused by magnetic correlations. Here, we report results from first principles calculations on the previously predicted theoretical collinear afm ground state that strongly support the presence of afm correlations in hexagonal close-packed iron. We show that anomalous splitting of the Raman mode can be explained by spin–phonon interactions. Moreover, we find that the calculated hyperfine field is very weak and would lead to hyperfine splitting below the resolution of Mössbauer experiments.