bluuebunny/crossref_metadata_2025 huggingface dataset:
The ecology of infectious disease: Effects of host diversity and community composition on Lyme disease risk
Authors: LoGiudice Kathleen , Ostfeld Richard S. , Schmidt Kenneth A. , Keesing Felicia
Published: January 2003

The extent to which the biodiversity and community composition of ecosystems affect their functions is an issue that grows ever more compelling as human impacts on ecosystems increase. We present evidence that supports a novel function of vertebrate biodiversity, the buffering of human risk of exposure to Lyme-disease-bearing ticks. We tested the Dilution Effect model, which predicts that high species diversity in the community of tick hosts reduces vector infection prevalence by diluting the effects of the most competent disease reservoir, the ubiquitous white-footed mouse (
Peromyscus leucopus
). As habitats are degraded by fragmentation or other anthropogenic forces, some members of the host community disappear. Thus, species-poor communities tend to have mice, but few other hosts, whereas species-rich communities have mice, plus many other potential hosts. We demonstrate that the most common nonmouse hosts are relatively poor reservoirs for the Lyme spirochete and should reduce the prevalence of the disease by feeding, but rarely infecting, ticks. By accounting for nearly every host species' contribution to the number of larval ticks fed and infected, we show that as new host species are added to a depauperate community, the nymphal infection prevalence, a key risk factor, declines. We identify important “dilution hosts” (e.g., squirrels), characterized by high tick burdens, low reservoir competence, and high population density, as well as “rescue hosts” (e.g., shrews), which are capable of maintaining high disease risk when mouse density is low. Our study suggests that the preservation of vertebrate biodiversity and community composition can reduce the incidence of Lyme disease.
Gα12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF
Authors: Suzuki Nobuchika , Nakamura Susumu , Mano Hiroyuki , Kozasa Tohru
Published: January 2003

Heterotrimeric G proteins, G12 and G13, have been shown to transduce signals from G protein-coupled receptors to activate Rho GTPase in cells. Recently, we identified p115RhoGEF, one of the guanine nucleotide exchange factors (GEFs) for Rho, as a direct link between Gα13 and Rho [Kozasa, T.,
et al.
(1998)
Science
280, 2109–2111; Hart, M. J.,
et al.
(1998)
Science
280, 2112–2114]. Activated Gα13 stimulated the RhoGEF activity of p115 through interaction with the N-terminal RGS domain. However, Gα12 could not activate Rho through p115, although it interacted with the RGS domain of p115. The biochemical mechanism from Gα12 to Rho activation remained unknown. In this study, we analyzed the interaction of leukemia-associated RhoGEF (LARG), which also contains RGS domain, with Gα12 and Gα13. RGS domain of LARG demonstrated Gα12- and Gα13-specific GAP activity. LARG synergistically stimulated SRF activation by Gα12 and Gα13 in HeLa cells, and the SRF activation by Gα12-LARG was further stimulated by coexpression of Tec tyrosine kinase. It was also found that LARG is phosphorylated on tyrosine by Tec. In reconstitution assays, the RhoGEF activity of nonphosphorylated LARG was stimulated by Gα13 but not Gα12. However, when LARG was phosphorylated by Tec, Gα12 effectively stimulated the RhoGEF activity of LARG. These results demonstrate the biochemical mechanism of Rho activation through Gα12 and that the regulation of RhoGEFs by heterotrimeric G proteins G12/13 is further modulated by tyrosine phosphorylation.
Urochordates and the origin of natural killer cells: Identification of a CD94/NKR-P1-related receptor in blood cells ofBotryllus
Authors: Khalturin Konstantin , Becker Matthias , Rinkevich Baruch , Bosch Thomas C. G.
Published: January 2003

Transplantation immunity based on the recognition of MHC molecules is well described in vertebrates. Vertebrates, however, do not undergo transplantation reaction naturally. The phylogenetically closest group in which transplantation reactions can occur is the Urochordata. Therefore, these animals occupy a key position for understanding the evolution of the vertebrate immune system. When screening for genes differentially expressed during allorecognition inBotryllus schlosseri, we isolated a gene coding for a type II transmembrane protein with a C-type lectin-binding domain and close similarity to vertebrates CD94 and NKR-P1. Here we show that the gene, BsCD94-1, is differentially regulated during allorecognition and that a subpopulation of blood cells carries the corresponding receptor on its cell surface. Southern blot analysis with DNA from individual colonies and intronless BsCD94-1probe reveal variation between individuals at the genomic level. CD94 in vertebrates is one of the markers for natural killer cells and binds to MHC class I molecules. Natural killer cells play a major role in recognition and elimination of allogeneic cells. Their evolutionary origin, however, remained unknown. The results presented here indicate that the elaboration of the vertebrate immune system may have its roots in an ancestral population of cells in the urochordate blood.
RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway
Authors: Suzumori Nobuhiro , Burns Kathleen H. , Yan Wei , Matzuk Martin M.
Published: January 2003

Oocyte meiosis and early mitotic divisions in developing embryos rely on the timely production of cell cycle regulators and their clearance via proteasomal degradation.
R
et
F
inger
P
rotein-
L
ike 4 (
Rfpl4
), encoding a RING finger-like protein with a B30.2 domain, was discovered during an
in silico
search for germ cell-specific genes. To study the expression and functions of RFPL4 protein, we performed immunolocalizations and used yeast two-hybrid and other protein–protein interaction assays. Immunohistochemistry and immunofluorescence showed that RFPL4 accumulates in all growing oocytes and quickly disappears during early embryonic cleavage. We used a yeast two-hybrid model to demonstrate that RFPL4 interacts with the E2 ubiquitin-conjugating enzyme HR6A, proteasome subunit β type 1, ubiquitin B, as well as a degradation target protein, cyclin B1. Coimmunoprecipitation analyses of
in vitro
translated proteins and extracts of transiently cotransfected Chinese hamster ovary (CHO)-K1 cells confirmed these findings. We conclude that, like many RING-finger containing proteins, RFPL4 is an E3 ubiquitin ligase. The specificity of its expression and these interactions suggest that RFPL4 targets cyclin B1 for proteasomal degradation, a key aspect of oocyte cell cycle control during meiosis and the crucial oocyte-to-embryo transition to mitosis.
Human Pumilio-2 is expressed in embryonic stem cells and germ cells and interacts with DAZ (Deleted in AZoospermia) and DAZ-Like proteins
Authors: Moore Frederick L. , Jaruzelska Jadwiga , Fox Mark S. , Urano Jun , Firpo Meri T. , Turek Paul J. , Dorfman David M. , Pera Renee A. Reijo
Published: January 2003

Early in development, a part of the embryo is set aside to become the germ cell lineage that will ultimately differentiate to form sperm and eggs and transmit genetic information to the next generation. Men with deletions encompassing the Y-chromosomeDAZgenes have few or no germ cells but are otherwise healthy, indicating they harbor specific defects in formation or maintenance of germ cells. ADAZhomolog,DAZL(DAZ-Like), is found in diverse organisms, including humans and is required for germ cell development in males and/or females. We identified proteins that interact with DAZ proteins to better understand their function in human germ cells. Here, we show that PUM2, a human homolog of Pumilio, a protein required to maintain germ line stem cells inDrosophilaandCaenorhabditis elegans, forms a stable complex with DAZ through the same functional domain required for RNA binding, protein–protein interactions and rescue ofPumiliomutations in flies. We also show thatPUM2is expressed predominantly in human embryonic stem cells and germ cells and colocalizes with DAZ and DAZL in germ cells. These data implicate PUM2 as a component of conserved cellular machinery that may be required for germ cell development.
Evidence that exposure of the telomere 3′ overhang sequence induces senescence
Authors: Li Guang-Zhi , Eller Mark S. , Firoozabadi Reza , Gilchrest Barbara A.
Published: January 2003

Normal human cells cease proliferation after a finite number of population doublings, a phenomenon termed replicative senescence. This process, first convincingly described by Hayflick and Moorhead [Hayflick, L. & Moorhead, P. S. (1961)
Exp. Cell Res.
25, 595–621] for cultured human fibroblasts 40 years ago, is suggested to be a fundamental defense against cancer. Several events have been demonstrated to induce the senescent phenotype including telomere shortening, DNA damage, oxidative stress, and oncogenic stimulation. The molecular mechanisms underlying senescence are poorly understood. Here we report that a 1-week exposure to oligonucleotide homologous to the telomere 3′-overhang sequence TTAGGG (T-oligo) similarly specifically induces a senescent phenotype in cultured human fibroblasts, mimicking serial passage or ectopic expression of a dominant negative form of the telomeric repeat binding factor, TRF2
DN
. We propose that exposure of the 3′ overhang due to telomere loop disruption may occur with critical telomere shortening or extensive acute DNA damage and that the exposed TTAGGG tandem repeat sequence then triggers DNA-damage responses. We further demonstrate that these responses can be induced by treatment with oligonucleotides homologous to the overhang in the absence of telomere disruption, a phenomenon of potential therapeutic importance.
A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes
Authors: Carlberg Inger , Hansson Maria , Kieselbach Thomas , Schröder Wolfgang P. , Andersson Bertil , Vener Alexander V.
Published: January 2003

The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have been unknown during two decades of studies on redox-dependent protein phosphorylation in plant photosynthetic membranes. Digestion of this protein from spinach thylakoid membranes with trypsin and subsequent tandem nanospray-quadrupole-time-of-flight mass spectrometry of the peptides revealed a protein sequence that did not correspond to any previously known protein. Sequencing of the corresponding cDNA uncovered a gene for a precursor protein with a transit peptide followed by a strongly basic mature protein with a molecular mass of 8,640 Da. Genes encoding homologous proteins were found on chromosome 3 of
Arabidopsis
and rice as well as in ESTs from 20 different plant species, but not from any other organisms. The protein can be released from the membrane with high salt and is also partially released in response to light-induced phosphorylation of thylakoids, in contrast to all other known thylakoid phosphoproteins, which are integral to the membrane. On the basis of its properties, this plant-specific protein is named thylakoid soluble phosphoprotein of 9 kDa (TSP9). Mass spectrometric analyses revealed the existence of non-, mono-, di-, and triphosphorylated forms of TSP9 and phosphorylation of three distinct threonine residues in the central part of the protein. The phosphorylation and release of TSP9 from the photosynthetic membrane on illumination favor participation of this basic protein in cell signaling and regulation of plant gene expression in response to changing light conditions.
Surface gas-exchange processes of snow algae
Authors: Williams William E. , Gorton Holly L. , Vogelmann Thomas C.
Published: January 2003

The red-colored chlorophyte
Chlamydomonas nivalis
is commonly found in summer snowfields. We used a modified Li-Cor gas-exchange system to investigate surface gas-exchange characteristics of snow colonized by this alga, finding rates of CO
2
uptake up to 0.3 μmol m
−2
⋅s
−1
in dense algal blooms. Experiments varying the irradiance resulted in light curves that resembled those of the leaves of higher plants. Red light was more effective than white and much more effective than green or blue, because of the red astaxanthin that surrounds and masks the algal chloroplasts. Integrating daily course measurements of gas exchange showed CO
2
uptake around 2,300 μmol⋅m
−2
⋅day
−1
in heavily colonized patches, indicating that summer snowfields can be surprisingly productive.
Sexual dimorphism in reduced-size liver ischemia and reperfusion injury in mice: Role of endothelial cell nitric oxide synthase
Authors: Harada Hirohisa , Pavlick Kevin P. , Hines Ian N. , Lefer David J. , Hoffman Jason M. , Bharwani Sulaiman , Wolf Robert E. , Grisham Matthew B.
Published: January 2003

We have recently reported that female mice are protected to a much greater extent from the injurious effects of reduced-size liver ischemia and reperfusion (RSL+I/R) than are males by an estrogen-dependent mechanism. The objective of this study was to examine the possibility that the protective effect observed in female mice depends on the up-regulation and/or activation of endothelial cell NO synthase (eNOS). Anesthetized female and male wild-type or eNOS-deficient C57BL/6 mice were subjected to 70% liver ischemia for 45 min followed by resection of the remaining 30% nonischemic lobes and reperfusion of ischemic tissue. Survival was monitored daily, whereas liver injury was quantified by using serum alanine aminotransferase determinations and histopathology. Hepatic eNOS mRNA, protein, and enzymatic activity were determined in male and female mice subjected to RSL+I/R. We found that liver injury was reduced and survival increased in female mice compared with males. This protective effect correlated with significant increases in hepatic eNOS message levels and enzyme activity but not protein expression compared with males subjected to the surgery. Furthermore,
N
ω
-nitro-
l
-arginine methyl ester-treated or eNOS-deficient female mice responded to RSL+I/R with dramatic increases in liver injury and 100% mortality within 2 days of surgery. Finally, we found that pravastatin pretreatment significantly attenuated hepatocellular injury and increased survival of male mice, which was associated with enhanced expression of eNOS message. We conclude that the protective effect afforded female mice is due to the activation of hepatic eNOS activity and enhanced NO production.
Cyclicity of mosquito vitellogenic ecdysteroid-mediated signaling is modulated by alternative dimerization of the RXR homologue
Ultraspiracle
Authors: Zhu Jinsong , Miura Ken , Chen Li , Raikhel Alexander S.
Published: January 2003

In anautogenous mosquitoes, egg maturation requires a blood meal. As a consequence, mosquitoes are vectors of numerous devastating human diseases. Blood feeding triggers a 20-hydroxyecdysone (20E) hormonal cascade, which activates yolk protein precursor (
YPP
) genes in the female fat body, an insect metabolic tissue. An important adaptation for anautogeny is the previtellogenic arrest preventing activation of
YPP
genes. Equally essential is termination of their expression, so that another arrest is achieved after a batch of eggs is laid. Here, we report that mosquito Seven-up (AaSvp), a chicken ovalbumin upstream promoter-transcription factor homologue, is involved in regulating the cyclicity of vitellogenic ecdysteroid-mediated signaling through heterodimerization with a retinoid X receptor homologue
Ultraspiracle
(USP), the obligatory functional ecdysteroid receptor (EcR) partner. AaSvp inhibits 20E-dependent activation of the vitellogenin (
Vg
) gene in transfection assays. Two-hybrid and GST pull-down analyses demonstrate that
in vitro
AaSvp interacts with both AaUSP and AaEcR. However, the coimmunoprecipitation using fat body nuclear extracts reveals that at 33–36 h postblood meal, when the 20E titer sharply declines and
YPP
gene expression ceases, AaSvp replaces AaEcR in USP heterodimers. The chromatin immunoprecipitation assay indicates that protein–protein interaction rather than binding competition for the
Vg
ecdysteroid response element accounts for the inhibition of
Vg
expression by AaSvp.
Rare earth-doped glass microbarcodes
Authors: Dejneka Matthew J. , Streltsov Alexander , Pal Santona , Frutos Anthony G. , Powell Christy L. , Yost Kevin , Yuen Po Ki , Müller Uwe , Lahiri Joydeep
Published: January 2003

The development of ultraminiaturized identification tags has applications in fields ranging from advanced biotechnology to security. This paper describes micrometer-sized glass barcodes containing a pattern of different fluorescent materials that are easily identified by using a UV lamp and an optical microscope. A model DNA hybridization assay using these “microbarcodes” is described. Rare earth-doped glasses were chosen because of their narrow emission bands, high quantum efficiencies, noninterference with common fluorescent labels, and inertness to most organic and aqueous solvents. These properties and the large number (>1 million) of possible combinations of these microbarcodes make them attractive for use in multiplexed bioassays and general encoding.
Exclusive tumor necrosis factor (TNF) signaling by the p75TNF receptor triggers inflammatory ischemia in the CNS of transgenic mice
Authors: Akassoglou Katerina , Douni Eleni , Bauer Jan , Lassmann Hans , Kollias George , Probert Lesley
Published: January 2003

Tumor necrosis factor (TNF) is up-regulated in a variety of central nervous system (CNS) diseases with diverse etiology and pathologic manifestation. TNF mediates multiple biological activities through two membrane receptors, the p55 and p75 TNF receptors (TNFRs). We have shown previously that human transmembrane TNF (tmTNF)/p55TNFR signaling in transgenic mice triggers oligodendrocyte apoptosis, endothelial cell activation, parenchymal inflammation, and primary demyelinating lesions similar to those of acute multiple sclerosis. To address the role of the p75TNFR in the CNS, we have generated “humanized” mice that express human tmTNF in astrocytes and a physiologically regulated human p75TNFR transgene, in the absence of the endogenous (murine) p55TNFR. Human tmTNF/p75TNFR transgenic mice develop CNS vascular pathology, characterized by endothelial cell activation, meningeal inflammation, and vessel fibrosis. There is no evidence of oligodendrocyte apoptosis or primary demyelination in these mice. Late in disease, vasculitis can result in vessel occlusion and secondary, multifocal CNS ischemic injury. These results identify a proinflammatory role for the p75TNFR at the level of the CNS vascular endothelium, which correlates with the expression pattern of this receptor in the CNS, and indicate that the differential expression patterns of the two TNFRs within the CNS play a significant role in shaping the outcome of TNF signaling during neuroimmune interactions.
Specificity of activation by phosphoinositides determines lipid regulation of Kir channels
Authors: Rohács Tibor , Lopes Coeli M. B. , Jin Taihao , Ramdya Pavan P. , Molnár Zoltán , Logothetis Diomedes E.
Published: January 2003

Phosphoinositides are critical regulators of ion channel and transporter activity. Defects in interactions of
i
nwardly
r
ectifying potassium (Kir) channels with phosphoinositides lead to disease. ATP-sensitive K
+
channels (K
ATP
) are unique among Kir channels in that they serve as metabolic sensors, inhibited by ATP while stimulated by long-chain (LC) acyl-CoA. Here we show that K
ATP
are the least specific Kir channels in their activation by phosphoinositides and we demonstrate that LC acyl-CoA activation of these channels depends on their low phosphoinositide specificity. We provide a systematic characterization of phosphoinositide specificity of the entire Kir channel family expressed in
Xenopus
oocytes and identify molecular determinants of such specificity. We show that mutations in the Kir2.1 channel decreasing phosphoinositide specificity allow activation by LC acyl-CoA. Our data demonstrate that differences in phosphoinositide specificity determine the modulation of Kir channel activity by distinct regulatory lipids.
Potent antitumor activity of a urokinase-activated engineered anthrax toxin
Authors: Liu Shihui , Aaronson Hannah , Mitola David J. , Leppla Stephen H. , Bugge Thomas H.
Published: January 2003

The acquisition of cell-surface urokinase plasminogen activator activity is a hallmark of malignancy. We generated an engineered anthrax toxin that is activated by cell-surface urokinase
in vivo
and displays limited toxicity to normal tissue but broad and potent tumoricidal activity. Native anthrax toxin protective antigen, when administered with a chimeric anthrax toxin lethal factor,
Pseudomonas exotoxin
fusion protein, was extremely toxic to mice, causing rapid and fatal organ damage. Replacing the furin activation sequence in anthrax toxin protective antigen with an artificial peptide sequence efficiently activated by urokinase greatly attenuated toxicity to mice. In addition, the mutation conferred cell-surface urokinase-dependent toxin activation
in vivo
, as determined by using a panel of plasminogen, plasminogen activator, plasminogen activator receptor, and plasminogen activator inhibitor-deficient mice. Surprisingly, toxin activation critically depended on both urokinase plasminogen activator receptor and plasminogen
in vivo
, showing that both proteins are essential cofactors for the generation of cell-surface urokinase. The engineered toxin displayed potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin and could eradicate established solid tumors. This tumoricidal activity depended strictly on tumor cell-surface plasminogen activation. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent.
Impaired B cell development and function in mice with a targeted disruption of the homeobox gene
Hex
Authors: Bogue Clifford W. , Zhang Ping-Xia , McGrath James , Jacobs Harris C. , Fuleihan Ramsay L.
Published: January 2003

Hex
is a homeobox gene that is expressed in all stages of B cell development except plasma cells. We studied lymphocyte development in the absence of
Hex
by using the
RAG1
-deficient blastocyst complementation system because homozygous disruption of
Hex
is embryonic lethal.
Hex
−/−
;
RAG1
−/−
chimeric mice had severely reduced numbers of mature B cells, pre-B cells, and CD5
+
B cells with a striking 15-fold increase in the percentage of B220
−
CD19
+
cells in the bone marrow.
Hex
−/−
;
RAG1
−/−
chimeric mice failed to generate IgG antibodies to T cell-independent antigens, although their serum IgM levels and antibody responses to T cell-dependent antigens were intact. Therefore,
Hex
is necessary for B cell development and function and its absence results in a dramatic increase in B220
−
CD19
+
cells.
High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years
Authors: Broxmeyer Hal E. , Srour Edward F. , Hangoc Giao , Cooper Scott , Anderson Stacie A. , Bodine David M.
Published: January 2003

Transplanted cord blood (CB) hematopoietic stem cells (HSC) and progenitor cells (HPC) can treat malignant and nonmalignant disorders. Because long-term cryopreservation is critical for CB banking and transplantation, we assessed the efficiency of recovery of viable HSC/HPC from individual CBs stored frozen for 15 yr. Average recoveries (± 1 SD) of defrosted nucleated cells, colony-forming unit-granulocyte, -macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, -erythrocyte, -monocyte, and -megakaryocyte (CFU-GEMM) were, respectively, 83 ± 12, 95 ± 16, 84 ± 25, and 85 ± 25 using the same culture conditions as for prefreeze samples. Proliferative capacities of CFU-GM, BFU-E, and CFU-GEMM were intact as colonies generated respectively contained up to 22,500, 182,500, and 292,500 cells. Self-renewal of CFU-GEMM was also retained as replating efficiency of single CFU-GEMM colonies into 2° dishes was >96% and yielded 2° colonies of CFU-GM, BFU-E, and CFU-GEMM. Moreover, CD34
+
CD38
−
cells isolated by FACS after thawing yielded >250-fold
ex vivo
expansion of HPC. To assess HSC capability, defrosts from single collections were bead-separated into CD34
+
cells and infused into sublethally irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. CD45
+
human cell engraftment with multilineage phenotypes was detected in mice after 11–13 wk; engrafting levels were comparable to that reported with fresh CB. Thus, immature human CB cells with high proliferative, replating,
ex vivo
expansion and mouse NOD/SCID engrafting ability can be stored frozen for >15 yr, can be efficiently retrieved, and most likely remain effective for clinical transplantation.
Expression and assembly of a fully active antibody in algae
Authors: Mayfield Stephen P. , Franklin Scott E. , Lerner Richard A.
Published: January 2003

Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga,
Chlamydomonas reinhardtii
. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two
C. reinhardtii
chloroplast promoters and 5′ and 3′ RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes
in vivo
. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5′ and 3′ elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.
Bimodal signal requisite for agonistic behavior in a dart-poison frog,Epipedobatesfemoralis
Authors: Narins Peter M. , Hödl Walter , Grabul Daniela S.
Published: January 2003

Animal acoustic signals play seminal roles in mate attraction and regulation of male spacing, maintenance of pairbonds, localization of hosts by parasites, and feeding behavior. Among vertebrate signals, it is becoming clear that no single stereotyped signal feature reliably elicits species-specific behavior, but rather, that a suite of characters is involved. Within the largely nocturnal clade of anuran amphibians, the dart-poison frog,Epipedobates femoralis, is a diurnal species that physically and vigorously defends its calling territory against conspecific intruders. Here we report that physical attacks by a territorial male are provoked only in response to dynamic bimodal stimuli in which the acoustic playback of vocalizations is coupled with vocal sac pulsations, but not by either unimodal cues presented in isolation or static bimodal stimuli. These results suggest that integration of dynamic bimodal cues is necessary to elicit aggression in this species.
The Euler–Maclaurin formula for simple integral polytopes
Authors: Karshon Yael , Sternberg Shlomo , Weitsman Jonathan
Published: January 2003

We give a Euler–Maclaurin formula with remainder for the sum of a smooth function on the integral points in a simple integral lattice polytope. Our proof uses elementary methods.
Dual roles of modulatory calcineurin-interacting protein 1 in cardiac hypertrophy
Authors: Vega Rick B. , Rothermel Beverly A. , Weinheimer Carla J. , Kovacs Atilla , Naseem R. H. , Bassel-Duby Rhonda , Williams R. S. , Olson Eric N.
Published: January 2003

The calcium/calmodulin-dependent protein phosphatase calcineurin stimulates cardiac hypertrophy in response to numerous stimuli. Calcineurin activity is suppressed by association with modulatory calcineurin-interacting protein (MCIP)1/DSCR1, which is up-regulated by calcineurin signaling and has been proposed to function in a negative feedback loop to modulate calcineurin activity. To investigate the involvement of MCIP1 in cardiac hypertrophy
in vivo
, we generated MCIP1 null mice and subjected them to a variety of stress stimuli that induce cardiac hypertrophy. In the absence of stress, MCIP1
−/−
animals exhibited no overt phenotype. However, the lack of MCIP1 exacerbated the hypertrophic response to activated calcineurin expressed from a muscle-specific transgene, consistent with a role of MCIP1 as a negative regulator of calcineurin signaling. Paradoxically, however, cardiac hypertrophy in response to pressure overload or chronic adrenergic stimulation was blunted in MCIP1
−/−
mice. These findings suggest that MCIP1 can facilitate or suppress cardiac calcineurin signaling depending on the nature of the hypertrophic stimulus. These opposing roles of MCIP have important implications for therapeutic strategies to regulate cardiac hypertrophy through modulation of calcineurin–MCIP activity.
Induction of neural crest in
Xenopus
by transcription factor AP2α
Authors: Luo Ting , Lee Young-Hoon , Saint-Jeannet Jean-Pierre , Sargent Thomas D.
Published: January 2003

We report experiments with
Xenopus laevis
, using both intact embryos and ectodermal explants, showing that the transcription factor AP2α is positively regulated by bone morphogenetic protein (BMP) and Wnt signaling, and that this activation is an essential step in the induction of neural crest (NC). Ectopic expression of AP2α is sufficient to activate high-level expression of NC-specific genes such as
Slug
and
Sox9
, which can occur as isolated domains within the neural plate as well as by expansion of endogenous NC territories. AP2α also has the property of inducing NC in isolated ectoderm in which Wnt signaling is provided but BMP signaling is minimized by overexpression of chordin. Like other NC regulatory factors, activation of
AP2α
requires some attenuation of endogenous BMP signaling; however, this process occurs at a lower threshold for
AP2α
. Furthermore,
AP2α
expression domains are larger than for other NC factors. Loss-of-function experiments with antisense AP2α morpholino oligonucleotides result in severe reduction in the NC territory. These results support a central role for AP2α in NC induction. We propose a model in which AP2α expression, along with inactivation of NC inhibitory factors such as Dlx3, establish a feedback loop comprising
AP2α
,
Sox9
, and
Slug
, leading to and maintaining NC specification.
Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase)
Authors: Miyadera Hiroko , Shiomi Kazuro , Ui Hideaki , Yamaguchi Yuichi , Masuma Rokuro , Tomoda Hiroshi , Miyoshi Hideto , Osanai Arihiro , Kita Kiyoshi , Ōmura Satoshi
Published: January 2003

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome
c
reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome
c
oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues.
Interstice mutations that block site-to-site translocation of a misactivated amino acid bound to a class I tRNA synthetase
Authors: Bishop Anthony C. , Beebe Kirk , Schimmel Paul R.
Published: January 2003

Class I aminoacyl-tRNA synthetases catalyze editing reactions that prevent ambiguity from entering the genetic code. Misactivated amino acids are translocated in cis from the active site for aminoacylation to the center for editing, located ≈30 Å away. Mutational analysis has functionally separated the two sites by creating mutations that disrupt the catalytic center for editing but not for aminoacylation and vice versa. What is not known is whether translocation
per se
can be disrupted without an effect on either catalytic center. Here we describe mutations in a presumptive “hinge region” of isoleucyl-tRNA synthetase that is situated between the two sites. Interstice mutations had little or no effect on either catalytic center. In contrast, the same specific mutations disrupted translocation. Thus, with these mutations all three functions, translocation, catalysis of aminoacylation, and editing, have been mutationally separated. The results are consistent with translocation involving a hinge-region conformational shift that does not perturb the two catalytic centers.
The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules
Authors: Lehtiö Janne , Sugiyama Junji , Gustavsson Malin , Fransson Linda , Linder Markus , Teeri Tuula T.
Published: January 2003

Cellulose binding modules (CBMs) potentiate the action of cellulolytic enzymes on insoluble substrates. Numerous studies have established that three aromatic residues on a CBM surface are needed for binding onto cellulose crystals and that tryptophans contribute to higher binding affinity than tyrosines. However, studies addressing the nature of CBM–cellulose interactions have so far failed to establish the binding site on cellulose crystals targeted by CBMs. In this study, the binding sites of CBMs on
Valonia
cellulose crystals have been visualized by transmission electron microscopy. Fusion of the CBMs with a modified staphylococcal protein A (ZZ-domain) allowed direct immuno-gold labeling at close proximity of the actual CBM binding site. The transmission electron microscopy images provide unequivocal evidence that the fungal family 1 CBMs as well as the family 3 CBM from
Clostridium thermocellum
CipA have defined binding sites on two opposite corners of
Valonia
cellulose crystals. In most samples these corners are worn to display significant area of the hydrophobic (110) plane, which thus constitutes the binding site for these CBMs.
Artificial cells: Unique insights into exocytosis using liposomes and lipid nanotubes
Authors: Cans Ann-Sofie , Wittenberg Nathan , Karlsson Roger , Sombers Leslie , Karlsson Mattias , Orwar Owe , Ewing Andrew
Published: January 2003

Exocytosis is the fundamental process underlying neuronal communication. This process involves fusion of a small neurotransmitter-containing vesicle with the plasma membrane of a cell to release minute amounts of transmitter molecules. Exocytosis is thought to go through an intermediate step involving formation of a small lipid nanotube or fusion pore, followed by expansion of the pore to the final stage of exocytosis. The process of exocytosis has been studied by various methods; however, when living cells are used it is difficult to discriminate between the molecular effects of membrane proteins relative to the mechanics of lipid–membrane-driven processes and to manipulate system parameters (e.g., membrane composition, pH, ion concentration, temperature, etc.). We describe the use of liposome–lipid nanotube networks to create an artificial cell model that undergoes the later stages of exocytosis. This model shows that membrane mechanics, without protein intervention, can drive expansion of the fusion pore to the final stage of exocytosis and can affect the rate of transmitter release through the fusion pore.
Quaternary structure of hemoglobin in solution
Authors: Lukin Jonathan A. , Kontaxis Georg , Simplaceanu Virgil , Yuan Yue , Bax Ad , Ho Chien
Published: January 2003

Many important proteins perform their physiological functions under allosteric control, whereby the binding of a ligand at a specific site influences the binding affinity at a different site. Allosteric regulation usually involves a switch in protein conformation upon ligand binding. The energies of the corresponding structures are comparable, and, therefore, the possibility that a structure determined by x-ray diffraction in the crystalline state is influenced by its intermolecular contacts, and thus differs from the solution structure, cannot be excluded. Here, we demonstrate that the quaternary structure of tetrameric human normal adult carbonmonoxy-hemoglobin can readily be determined in solution at near-physiological conditions of pH, ionic strength, and temperature by NMR measurement of
15
N-
1
H residual dipolar couplings in weakly oriented samples. The structure is found to be a dynamic intermediate between two previously solved crystal structures, known as the R and R2 states. Exchange broadening at the subunit interface points to a rapid equilibrium between different structures that presumably include the crystallographically observed states.
The solitary wave of asexual evolution
Authors: Rouzine Igor M. , Wakeley John , Coffin John M.
Published: January 2003

Using a previously undescribed approach, we develop an analytic model that predicts whether an asexual population accumulates advantageous or deleterious mutations over time and the rate at which either process occurs. The model considers a large number of linked identical loci, or nucleotide sites; assumes that the selection coefficient per site is much less than the mutation rate per genome; and includes back and compensating mutations. Using analysis and Monte Carlo simulations, we demonstrate the accuracy of our results over almost the entire range of population sizes. Two limiting cases of our results, when either deleterious or advantageous mutations can be neglected, correspond to the Fisher–Muller effect and Muller's ratchet, respectively. By comparing predictions of our model (no recombination) to those of simple single-locus models (strong recombination), we show that the accumulation of advantageous mutations is slowed by linkage over a broad, finite range of population size. This supports the view of Fisher and Muller, who argued in the 1930s that progressive evolution of organisms is slowed because loci at which beneficial mutations can occur are often linked together on the same chromosome. These results follow from our main finding, that distribution of sequences over the mutation number evolves as a traveling wave whose speed and width depend on population size and other parameters. The model explains a logarithmic dependence of steady-state fitness on the population size reported recently for an RNA virus.
Chaperones increase association of tau protein with microtubules
Authors: Dou Fei , Netzer William J. , Tanemura Kentaro , Li Feng , Hartl F. Ulrich , Takashima Akihiko , Gouras Gunnar K. , Greengard Paul , Xu Huaxi
Published: January 2003

Molecular chaperones and their functions in protein folding have been implicated in several neurodegenerative diseases, including Parkinson's disease and Huntington's disease, which are characterized by accumulation of protein aggregates (e.g., α-synuclein and huntingtin, respectively). These aggregates have been shown in various experimental systems to respond to changes in levels of molecular chaperones suggesting the possibility of therapeutic intervention and a role for chaperones in disease pathogenesis. It remains unclear whether chaperones also play a role in Alzheimer's disease, a neurodegenerative disorder characterized by β-amyloid and tau protein aggregates. Here, we report an inverse relationship between aggregated tau and the levels of heat shock protein (Hsp)70/90 in tau transgenic mouse and Alzheimer's disease brains. In various cellular models, increased levels of Hsp70 and Hsp90 promote tau solubility and tau binding to microtubules, reduce insoluble tau and cause reduced tau phosphorylation. Conversely, lowered levels of Hsp70 and Hsp90 result in the opposite effects. We have also demonstrated a direct association of the chaperones with tau proteins. Our results suggest that up-regulation of molecular chaperones may suppress formation of neurofibrillary tangles by partitioning tau into a productive folding pathway and thereby preventing tau aggregation.
Mutations linked to dyskeratosis congenita cause changes in the structural equilibrium in telomerase RNA
Authors: Theimer Carla A. , Finger L. David , Trantirek Lukas , Feigon Juli
Published: January 2003

Autosomal dominant dyskeratosis congenita (DKC), as well as aplastic anemia, has been linked to mutations in the RNA component of telomerase, the ribonucleoprotein responsible for telomere maintenance. Here we examine the effect of the DKC mutations on the structure and stability of human telomerase RNA pseudoknot and CR7 domains by using NMR and thermal melting. The CR7 domain point mutation decreases stability and alters a conserved secondary structure thought to be involved in human telomerase RNA accumulation
in vivo
. We find that pseudoknot constructs containing the conserved elements of the pseudoknot domain are in equilibrium with a hairpin conformation. The solution structure of the wild-type hairpin reveals that it forms a continuous helix containing a novel run of three consecutive U⋅U and a U⋅C base pairs closed by a pentaloop. The six base pairs unique to the hairpin conformation are phylogenetically conserved in mammals, suggesting that this conformation is also functionally important. The DKC mutation in the pseudoknot domain results in a shift in the equilibrium toward the hairpin form, primarily due to destabilization of the pseudoknot. Our results provide insight into the effect of these mutations on telomerase structure and suggest that the catalytic cycle of telomerase involves a delicate interplay between RNA conformational states, alteration of which leads to the disease state.
Permissive corridor and diffusible gradients direct medial ganglionic eminence cell migration to the neocortex
Authors: Wichterle Hynek , Alvarez-Dolado Manuel , Erskine Lynda , Alvarez-Buylla Arturo
Published: January 2003

Young neurons born in the medial ganglionic eminence (MGE) migrate a long distance dorsally, giving rise to several types of interneurons in neocortex. The mechanisms that facilitate selective dorsal dispersion of MGE cells while restricting their movement ventrally into neighboring regions are not known. Using microtransplantation into fetal brain slices and onto dissociated substrate cells on floating filters (spot assay), we demonstrate that ventral forebrain regions neighboring the MGE are nonpermissive for MGE cell migration, whereas the dorsal regions leading to the neocortex are increasingly permissive. Spot assay experiments using filters with different pore sizes indicate that the permissive factors are not diffusible. We also show that MGE cells respond to chemoattractive and inhibitory factors diffusing from the neocortex and ventromedial forebrain, respectively. We propose that the final extent and regional specificity of MGE cell dispersion is largely dictated by contact guidance through a selectively permissive environment, flanked by nonpermissive tissues. In addition, we propose that chemotactic guidance cues superimposed over the permissive corridor facilitate efficient dorsal migration of MGE cells.
WNK1, a kinase mutated in inherited hypertension with hyperkalemia, localizes to diverse Cl
−
-transporting epithelia
Authors: Choate Keith A. , Kahle Kristopher T. , Wilson Frederick H. , Nelson-Williams Carol , Lifton Richard P.
Published: January 2003

Mutations in
WNK1
and
WNK4
, genes encoding members of a novel family of serine–threonine kinases, have recently been shown to cause pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder featuring hypertension, hyperkalemia, and renal tubular acidosis. The localization of these kinases in the distal nephron and the Cl
−
dependence of these phenotypes suggest that these mutations increase renal Cl
−
reabsorption. Although
WNK4
expression is limited to the kidney,
WNK1
is expressed in many tissues. We have examined the distribution of
WNK1
in these extrarenal tissues. Immunostaining using WNK1-specific antibodies demonstrated that WNK1 is not present in all cell types; rather, it is predominantly localized in polarized epithelia, including those lining the lumen of the hepatic biliary ducts, pancreatic ducts, epididymis, sweat ducts, colonic crypts, and gallbladder. WNK1 is also found in the basal layers of epidermis and throughout the esophageal epithelium. The subcellular localization of WNK1 varies among these epithelia. WNK1 is cytoplasmic in kidney, colon, gallbladder, sweat duct, skin, and esophagus; in contrast, it localizes to the lateral membrane in bile ducts, pancreatic ducts, and epididymis. These epithelia are all notable for their prominent role in Cl
−
flux. Moreover, these sites largely coincide with those involved in the pathology of cystic fibrosis, a disease characterized by deranged epithelial Cl
−
flux. Together with the known pathophysiology of PHAII, these findings suggest that WNK1 plays a general role in the regulation of epithelial Cl
−
flux, a finding that suggests the potential of new approaches to the selective modulation of these processes.
Photocleavage of a 2-nitrobenzyl linker bridging a fluorophore to the 5′ end of DNA
Authors: Bai Xiaopeng , Li Zengmin , Jockusch Steffen , Turro Nicholas J. , Ju Jingyue
Published: January 2003

Three single-stranded DNA molecules of different lengths were synthesized and characterized, each containing a fluorescent dye (6-carboxyfluorescein) connected to the 5′ end via a photocleavable 2-nitrobenzyl linker and a biotin moiety at the 3′ end. UV irradiation (λ ≈ 340 nm) of solutions containing these fluorescent DNA molecules caused the complete cleavage of the nitrobenzyl linker, separating the fluorophore from the DNA. The photocleavage products were characterized by HPLC and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Our experimental results indicated that the proximity of the chromophore 6-carboxyfluorescein to the 2-nitrobenzyl linker did not hinder the quantitative photocleavage of the linker in the DNA molecules. The biotin moiety allowed immobilization of the fluorescent DNA on streptavidin-coated glass chips. The photocleavage of the immobilized DNA was investigated directly by fluorescence spectroscopy. The results demonstrated that close to 80% of the fluorophore was removed from the immobilized DNA after UV irradiation at 340 nm. These results strongly support the application of the 2-nitrobenzyl moiety as an efficient photocleavable linker, connecting fluorescent probes to DNA molecules for a variety of biological analyses such as DNA sequencing by synthesis.