bluuebunny/crossref_metadata_2025 huggingface dataset:
42 DEVELOPMENT OF PIG EMBRYOS CLONED FROM DONOR CELLS TREATED WITH TRICHOSTATIN A
Authors: Li J. , Du Y. , Kragh P. M. , Purup S. , Villemoes K. , Pedersen A. M. , Jørgensen A. L. , Bolund L. , Yang H. M. , Vajta G.
Published: January 2008

Development to the blastocyst stage following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to a totipotent state. Reprogramming of the transferred somatic nuclei must be completed by the time normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, Enright et al. (2003 Biol. Reprod. 69, 896–901) reported that in vitro development of cloned cow embryos was improved by treatment of donor cells with a histone deacetylase inhibitor, TrichostatinA (TSA). So far, there are no reports available for adult pig fibroblast cells treated with TSA. The objective of this study was to investigate whether the development of handmade cloned embryos in pig could be improved by using TSA-treated donor cells. Adult pig fibroblast cells were treated with 100, 150, or 200 nm TSA for 24 h, compared to untreated controls, and were then used as donor cells. The cells were electrofused with handmade enucleated pig oocytes separately and were activated with calcium ionophore and cycloheximide. They were subsequently cultured in porcine zygote medium 3 (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) using the well of the well system (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Experiments were repeated 4 times and the data were analyzed with AVEDEV and t-test in Excel (Microsoft Excel 2007). The cleavage rates and the total cell numbers per blastocyst were similar between groups (P > 0.05), as shown in Table 1. However, the cloned blastocyst rate using donor cells treated with 100 nm TSA was higher than in the other groups (69.9 ± 4.7% v. 43.6 ± 4.3%, 43.1 ± 5.8%, or 46.6 ± 3.6%; P < 0.05), as shown in Table 1. These data suggest that proper TSA treatment for donor cells before somatic cloning improves the rate of development of porcine handmade cloned embryos to the blastocyst stage. Further research is needed to examine the in vivo development of embryos reconstructed with TSA-treated donor cells.

Table 1. Developmental ability of cloned pig embryos derived fromTSA-treated donor cells
58 PRODUCTION OF CLONED BOVINE EMBRYOS DERIVED FROM AMNIOTIC CELLS OF PREGNANT COWS
Authors: Taniguchi S. , Hayashi N. , Abe Y. , Iwamoto D. , Kishigami S. , Kishi M. , Kato H. , Mitani T. , Matsumoto K. , Hosoi Y. , Iritani A. , Nagao Y. , Saeki K.
Published: January 2008

Progeny tests are widely used for selection of sires for beef and dairy cattle. A less costly method might be to clone the sire candidates at their earliest developmental stage possible. To produce cloned bulls, we obtained amniotic cells as donors for nuclear transfer by transvaginal aspiration of pregnant cows. However, the collected cells may include some maternal cells. In this study, we examined collection methods to obtain only fetal cells from the collected fluid. We also examined the developmental capacity of the embryos cloned from these cells. Amniotic fluids were aspirated from pregnant cows by ultrasound-guided aspiration. We collected amniotic fluids from 27 pregnant Japanese black beef cattle (between 58 and 132 days of gestation). In Method 1, cells were recovered from the whole amniotic fluid (approximately 15 mL). In Method 2, the initial 5 mL of aspirated fluid was discarded and then the next 10 mL sample was collected. Cells were recovered from the collected fluids. The cells in the fluids were washed twice by centrifugation and then cultured in AmnioMAX-II medium (GIBCO, Grand Island, NY, USA). After 3–4 passages, the sex of the cell lines was determined by the loop-mediated isothermal amplification (LAMP) method (Eiken Chemical Co., Ltd., Tokyo, Japan). For the cell lines that were determined as 'male' by the LAMP method we further analyzed the sex of individual cells (137–620 cells of each cell line) by fluorescent in situ hybridization (FISH) using a bovineY chromosome-specific probe (Kobayashi et al. 1998 Mol. Reprod. Dev. 51, 390–394). The percentage of male cells obtained from Methods 1 and 2 were 0–0.4% (from 4 animals) and 93.7–99.5% (from 6 animals), respectively. Then, we used confluent amniotic cells from 3 cell lines obtained by Method 2 as donor cells for nuclear transfer and examined the developmental capacity of the cloned embryos. Bovine fibroblasts cultured under serum starvation were used as a control. The cells were electrically fused (2.7 kV cm–1, 11 µs, 2 times) with enucleated bovine oocytes, and activated with a calcium ionophore and cycloheximide. They were subsequently cultured in mSOF until 168 h post-activation. The data were analyzed with Fisher's protected least-squares difference (PLSD) test following ANOVA. The rates of fusion, cleavage, and development to the blastocyst stage of the cloned embryos were the same as those of the control embryos (78% v. 81%, 75% v. 75%, and 22% v. 27%, respectively; P > 0.05). Furthermore, the rate of male blastocysts derived from the cloned embryos with the three cell lines was 95% (19/20). These results indicate that the amniotic fluids collected from pregnant cows by Method 2 contained fewer maternal cells, and that the embryos cloned from the cells developed in a manner similar to that of embryos cloned from the fibroblasts.
This work was supported byWakayama Prefecture CREATE, JST.
63 PRODUCTION OF CLONED TRANSGENIC RED CATS
Authors: Yin X. J. , Lee H. S. , Yu X. F. , Choi E. G. , Koo B. C. , Kwon M. S. , Lee Y. S. , Cho S. J. , Jin G. Z. , Kim L. H. , Shin H. D. , Kim T. , Kim N. H. , Kong I. K.
Published: January 2008

A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Because the feline gene map displays a higher level of systemic conservation with the human genome than with rodents or other laboratory mammals, cats may offer a more appropriate model in which to study potential therapies for human diseases. A second point of application is the production of transgenic cats, genetically engineered designer pets with favorable attributes, such as allergen-free cats. Mature oocytes from superovulated cat ovaries were collected. Donor fibroblasts were obtained from an ear-skin biopsy of a white maleTurkish Angora cat, cultured for one to two passages, and then subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. Single donor cells emitting red fluorescence were selected under an inverted microscope equipped with an RFP filter set. For somatic cell nuclear transfer (SCNT), denuded oocytes were treated with 0.2 µg m L–1 demecolcine for 1 h, and the protruding first polar body and chromatin plate were removed by micromanipulation. A single donor cell was transferred into each enucleated oocyte. In total, 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 � 7.5 per recipient). Three of eleven surrogate mothers (S) were successfully impregnated (27.3%). Surrogate S-2 delivered one liveborn and one stillborn kitten at 65 days of gestation while S-3 delivered one liveborn kitten at 66 days of gestation. Microsatellite analysis revealed that all kittens were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cats, but not in the non-TG cats under excitation emission filter sets. The expression pattern of the RFP transgene was determined in several tissues taken from TG stillborn and non-TG cats. Although expression in the heart was detected more weakly than in other organs, red fluorescence was present in all tissue samples tested, but not in those from the non-TG cat. This is the first report of the successful production of a cloned transgenic cat expressing an exogenous gene. Our nuclear transfer procedure using genetically modified somatic cells would be useful for the efficient production of transgenic cats. The application of the SCNT procedure to produce genetically modified cats would be valuable for biomedical modeling of human disease, as well as for the production of designer pets.
This work was supported by a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MOST) (grant # M10525010001-05N2501-00110).
65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE
Authors: Zhou H. M. , Li B. S. , Zhang L. J.
Published: January 2008

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts.

Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed embryos
79 DEVELOPMENT TO TERM OF RAT ZYGOTES DERIVED FROM CRYOPRESERVED MATURE OOCYTES AND SPERM THROUGH INTRACYTOPLASMIC SPERM INJECTION
Authors: Kashiwazaki N. , Sano D. , Seita Y. , Sugio S. , Suzukamo C. , Nakata M. , Furugaichi A. , Hoshina M. , Inomata T. , Ito J.
Published: January 2008

The rat, as well as the mouse, is one of the most valuable experimental animals for biomedical and physiological research. There are numerous valuable mutant rats including transgenetic strains. Cryopreservation of rat oocytes and sperm as haploid germ cells is a key technology for banking the genetic resources efficiently. The aim of the present study was to examine survival of vitrified/warmed oocytes and developmental competence of resultant zygotes in the rat. Rats used in the present study were all Wistar rats. Epididymal spermatozoa were frozen as described previously (Seita et al. 2005 Reprod. Fertil. Dev. 18, 256). After thawing, spermatozoa were sonicated to obtain sperm heads for intracytoplasmic sperm injection (ICSI). Oocytes were collected from immature females superovulated with eCG and hCG. Oocytes were equilibrated in 7.5% (v/v) ethylene glycol (EG) + 7.5% (v/v) dimethylsulfoxide (DMSO) + 20% (v/v) FCS in PB1 for 5 min and then transferred into 15.0% EG (v/v) + 15.0% DMSO (v/v) + 20% FCS + 0.5 m sucrose in PB1 (vitrification solution) for 1 min at room temperature (22–24�C). During exposure to the vitrification solution, oocytes were loaded on a Cryotop� (Kitazato Supply Co., Tokyo, Japan). At warming, the film of Cryotop was directly immersed into PB1 containing 0.5 m sucrose and 20% FCS at 37.5�C. The warmed oocytes were washed three times and put into a HEPES-buffered (22 mm) modified R1ECM (310 mOsm) medium. The sperm heads were microinjected intracytoplasmically into the warmed oocytes. Then, presumptive zygotes were transferred surgically into the oviducts of recipient females (Day 0), and Caesarean section of the recipients was performed on Day 22. After vitrification and warming, 245 of 275 (88%) oocytes survived morphologically, 240 of the warmed oocytes were injected, and 156 oocytes (65%) were morphologically normal after the injection. To confirm development to term of zygotes derived from cryopreserved oocytes and sperm, 143 injected oocytes were transferred to 9 recipients, resulting in 3 pregnancies and the generation of one live pup. The results indicate that rat zygotes derived from cryopreserved oocytes and sperm through ICSI can develop to term, and full developmental competence can be preserved in rat oocytes after cryopreservation.
80 EFFECT OF PRE-EQUILIBRATION TIME ON THE SURVIVAL RATE OF IN VITRO-FERTILIZED BOVINE EMBRYOS AFTER VITRIFICATION
Authors: Koyama H. , Sugulle A. H. , Dochi O.
Published: January 2008

Successful cryopreservation of embryos depends on the pre-equilibration time. This study was designed to compare 2 pre-equilibration times – short (1 min) and long (5 min) – and to evaluate the re-expansion and hatching rates of different stages of embryos using the short pre-equilibration method. Cumulus–oocyte complexes (COCs) from ovaries obtained from a slaughterhouse were matured, fertilized, and cultured in vitro. In Experiment 1, expanded blastocysts between 7 and 9 days of culture were pre-equilibrated for the short time (1 min) in 100 μL of vitrification solution 1 (VS1: containing 7.5% ethylene glycol (EG), 7.5% dimethyl sulfoxide (DMSO), and 20% calf serum (CS) in TCM-199), followed by incubation in 100 μL of vitrification solution 2 (VS2: containing 15% EG, 15% DMSO, 20% CS, and 1 m sucrose (Suc) in TCM-199) for 30 s. Another group of blastocysts was subjected to the long pre-equilibration (5 min) in 100 μL VS1, followed by incubation in 100 μL of VS2 for 30 s. The embryos were placed into Cryotops® (Kitasato Supply Co., Tokyo, Japan) and immediately submerged in liquid nitrogen and kept there for 1 week. Blastocysts were warmed by plunging the Cryotops into 1 m Suc in TCM-199 for 1 min, placed in 0.5 m Suc in TCM-199 for 3 min, and finally placed in CR1aa alone for 5 min before being cultured. In Experiment 2, 8-cell embryos, morulae, and expanded blastocysts were vitrified by the previously described short equilibration method. The re-expansion and hatching rates of embryos were determined as the percentage of vitrified–warmed embryos undergoing further development in the in vitro culture. The data were analyzed by the chi-square test. Results are presented in Table 1. There was no difference between the short and long pre-equilibration times in terms of survival (94.0% v. 94.1%, respectively) and morphological appearance immediately after warming. However, re-expansion of blastocysts (ability to develop further) was slightly higher with the short pre-equilibration than with the long pre-equilibration (90.0% v. 85.9%, respectively). In Experiment 2, there were no differences in embryo re-expansion, but the hatching rates of 8-cell embryos were lower than those of morulae, blastocysts, and controls. In conclusion, the results of this study indicate that the length of pre-equilibration time prior to vitrification does not influence embryo re-expansion, and that bovine morulae and blastocysts can be vitrified with equal success. We also conclude that insufficient permeation of cryoprotectants may occur in 8-cell embryos.

Table 1. Survival and hatching rates of vitrified bovine embryos after warming
Signal processing by its coil zipper domain activates IKKγ
Authors: Bloor Stuart , Ryzhakov Grigor , Wagner Sebastian , Butler P. Jonathan G. , Smith David L. , Krumbach Rebekka , Dikic Ivan , Randow Felix
Published: January 2008

NF-κB activation occurs upon degradation of its inhibitor I-κB and requires prior phosphorylation of the inhibitor by I-κB kinase (IKK). Activity of IKK is governed by its noncatalytic subunit IKKγ. Signaling defects due to missense mutations in IKKγ have been correlated to its inability to either become ubiquitylated or bind ubiquitin noncovalently. Because the relative contribution of these events to signaling had remained unknown, we have studied mutations in the coil-zipper (CoZi) domain of IKKγ that either impair signaling or cause constitutive NF-κB activity. Certain signaling-deficient alleles neither bound ubiquitin nor were they ubiquitylated by TRAF6. Introducing an activating mutation into those signaling-impaired alleles restored their ubiquitylation and created mutants constitutively activating NF-κB without repairing the ubiquitin-binding defect. Constitutive activity therefore arises downstream of ubiquitin binding but upstream of ubiquitylation. Such constitutive activity reveals a signal-processing function for IKKγ beyond that of a mere ubiquitin-binding adaptor. We propose that this signal processing may involve homophilic CoZi interactions as suggested by the enhanced affinity of CoZi domains from constitutively active IKKγ.
How subunit coupling produces the γ-subunit rotary motion in F
1
-ATPase
Authors: Pu Jingzhi , Karplus Martin
Published: January 2008

F
o
F
1
-ATP synthase manufactures the energy “currency,” ATP, of living cells. The soluble F
1
portion, called F
1
-ATPase, can act as a rotary motor, with ATP binding, hydrolysis, and product release, inducing a torque on the γ-subunit. A coarse-grained plastic network model is used to show at a residue level of detail how the conformational changes of the catalytic β-subunits act on the γ-subunit through repulsive van der Waals interactions to generate a torque that drives unidirectional rotation, as observed experimentally. The simulations suggest that the calculated 85° substep rotation is driven primarily by ATP binding and that the subsequent 35° substep rotation is produced by product release from one β-subunit and a concomitant binding pocket expansion of another β-subunit. The results of the simulation agree with single-molecule experiments [see, for example, Adachi K,
et al.
(2007)
Cell
130:309–321] and support a tri-site rotary mechanism for F
1
-ATPase under physiological condition.
Efficient coupling of catalysis and dynamics in the E1 component of
Escherichia coli
pyruvate dehydrogenase multienzyme complex
Authors: Kale Sachin , Ulas Gözde , Song Jaeyoung , Brudvig Gary W. , Furey William , Jordan Frank
Published: January 2008

Protein motions are ubiquitous and are intrinsically coupled to catalysis. Their specific roles, however, remain largely elusive. Dynamic loops at the active center of the E1 component of
Escherichia coli
pyruvate dehydrogenase multienzyme complex are essential for several catalytic functions starting from a predecarboxylation event and culminating in transfer of the acetyl moiety to the E2 component. Monitoring the kinetics of E1 and its loop variants at various solution viscosities, we show that the rate of a chemical step is modulated by loop dynamics. A cysteine-free E1 construct was site-specifically labeled on the inner loop (residues 401–413), and the EPR nitroxide label revealed ligand-induced conformational dynamics of the loop and a slow “open ↔ close” conformational equilibrium in the unliganded state. An
19
F NMR label placed at the same residue revealed motion on the millisecond-second time scale and suggested a quantitative correlation of E1 catalysis and loop dynamics for the 200,000-Da protein. Thermodynamic studies revealed that these motions may promote covalent addition of substrate to the enzyme-bound thiamin diphosphate by reducing the free energy of activation. Furthermore, the global dynamics of E1 presumably regulate and streamline the catalytic steps of the overall complex by inducing an entirely entropic (nonmechanical) negative cooperativity with respect to substrate binding at higher temperatures. Our results are consistent with, and reinforce the hypothesis of, coupling of catalysis and regulation with enzyme dynamics and suggest the mechanism by which it is achieved in a key branchpoint enzyme in sugar metabolism.
A
Caenorhabditis elegans
allatostatin/galanin-like receptor NPR-9 inhibits local search behavior in response to feeding cues
Authors: Bendena William G. , Boudreau Jeff R. , Papanicolaou Tony , Maltby Matt , Tobe Stephen S. , Chin-Sang Ian D.
Published: January 2008

Movement in
Caenorhabditis elegans
is the result of sensory cues creating stimulatory and inhibitory output from sensory neurons. Four interneurons (AIA, AIB, AIY, and AIZ) are the primary recipients of this information that is further processed en route to motor neurons and muscle contraction.
C. elegans
has >1,000 G protein-coupled receptors (GPCRs), and their contribution to sensory-based movement is largely undefined. We show that an allatostatin/galanin-like GPCR (NPR-9) is found exclusively in the paired AIB interneuron. AIB interneurons are associated with local search/pivoting behavior.
npr
-9 mutants display an increased local search/pivoting that impairs their ability to roam and travel long distances on food. With impaired roaming behavior on food
npr-9
mutants accumulate more intestinal fat as compared with wild type. Overexpression of NPR-9 resulted in a gain-of-function phenotype that exhibits enhanced forward movement with lost pivoting behavior off food. As such the animal travels a great distance off food, creating arcs to return to food. These findings indicate that NPR-9 has inhibitory effects on the AIB interneuron to regulate foraging behavior, which, in turn, may affect metabolic rate and lipid storage.
Genomic evidence for independent origins of β-like globin genes in monotremes and therian mammals
Authors: Opazo Juan C. , Hoffmann Federico G. , Storz Jay F.
Published: January 2008

Phylogenetic reconstructions of the β-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult β-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic β-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the “ε-globin” gene in birds is not orthologous to the ε-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto β-globin gene. Here, we report evidence that the early and late expressed β-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto β-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the ε- and β-globin genes of therian mammals arose via duplication of a proto β-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of β-like globin genes that originated via duplication of a proto β-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto β-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development.
Transient regulation of transport by pericytes in venular microvessels via trapped microdomains
Authors: Zhang X. , Adamson R. H. , Curry F. E. , Weinbaum S.
Published: January 2008

A phenomenon that has defied explanation for two decades is the time scale for transient reabsorption in the classic experiments of Michel and Phillips on individually perfused frog mesentery microvessels. One finds that transient reabsorption lasts <2 min before a new steady state of low filtration is established when the lumen pressure is abruptly dropped from a high to a low value. Our experiments in frog and rat venular microvessels under a variety of conditions revealed the same time trend for new steady states to be established as in Michel and Phillips' experiments. In contrast, one theoretically predicts herein that the time required for the tissue albumin concentration to increase to values for a new steady state to be achieved through reabsorption is in the order of several hours. In this paper we propose a new hypothesis and theoretical model for this rapid regulation, namely that pericytes covering the interendothelial cleft exits create small trapped microdomains outside the cleft exits which regulate this transient behavior. Our electron microscopy studies on rat mesenteric venular microvessels reveal an average pericyte coverage of ≈85%. The theoretical model based on this ultrastructural study predicts an equilibration time on the order of 1 min when the lumen pressure is abruptly lowered. The basic concept of a trapped microdomain can also be extended to microdomains in the interstitial space surrounding skeletal muscle capillaries.
Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured
Arabidopsis
cells
Authors: Pauwels Laurens , Morreel Kris , De Witte Emilie , Lammertyn Freya , Van Montagu Marc , Boerjan Wout , Inzé Dirk , Goossens Alain
Published: January 2008

Jasmonates (JAs) are plant-specific signaling molecules that steer a diverse set of physiological and developmental processes. Pathogen attack and wounding inflicted by herbivores induce the biosynthesis of these hormones, triggering defense responses both locally and systemically. We report on alterations in the transcriptome of a fast-dividing cell culture of the model plant
Arabidopsis thaliana
after exogenous application of methyl JA (MeJA). Early MeJA response genes encoded the JA biosynthesis pathway proteins and key regulators of MeJA responses, including most JA ZIM domain proteins and MYC2, together with transcriptional regulators with potential, but yet unknown, functions in MeJA signaling. In a second transcriptional wave, MeJA reprogrammed cellular metabolism and cell cycle progression. Up-regulation of the monolignol biosynthesis gene set resulted in an increased production of monolignols and oligolignols, the building blocks of lignin. Simultaneously, MeJA repressed activation of M-phase genes, arresting the cell cycle in G
2
. MeJA-responsive transcription factors were screened for their involvement in early signaling events, in particular the regulation of JA biosynthesis. Parallel screens based on yeast one-hybrid and transient transactivation assays identified both positive (MYC2 and the AP2/ERF factor ORA47) and negative (the C2H2 Zn finger proteins STZ/ZAT10 and AZF2) regulators, revealing a complex control of the JA autoregulatory loop and possibly other MeJA-mediated downstream processes.
Biologically templated organic polymers with nanoscale order
Authors: Willis Bert , Eubanks Lisa M. , Wood Malcolm R. , Janda Kim D. , Dickerson Tobin J. , Lerner Richard A.
Published: January 2008

Methods for the construction of ordered nanoscale arrays have been implicated in fields ranging from separation technologies to microelectronics. Yet, despite the plethora of nanoscale structures assembled in nature that use a templating strategy, chemists have been unable to replicate this success. A technology is reported for templated organic polymers composed of filamentous bacteriophage-polyacrylamide biomacromolecules that self-assemble into highly ordered helical bundles displaying hexagonal close packing. The results align with a previously reported mathematical prediction for the close packing of flexible tubes. This biopolymeric assembly can be viewed as a magnification of the inherent microscopic chirality and helicity present in individual phage particles at the macroscale level.
Heat-shock protein 90 associates with N-terminal extended peptides and is required for direct and indirect antigen presentation
Authors: Callahan Margaret K. , Garg Manish , Srivastava Pramod K.
Published: January 2008

CD8+T cells recognize peptide fragments of endogenously synthesized antigens of cancers or viruses, presented by MHC I molecules. Such antigen presentation requires the generation of peptides in the cytosol, their passage to the endoplasmic reticulum, loading of MHC I with peptides, and transport of MHC I–peptide complexes to the cell surface. Heat-shock protein (hsp) 90 is a cytosolic chaperone known to associate with peptide and peptide precursors of MHC I epitopes. We report here that treatment of cells with hsp90 inhibitors leads to generation of “empty” MHC I caused by inhibited loading of MHC I with peptides. Inhibition of hsp90 does not inhibit synthesis of MHC I, nor does it affect the activity of proteasomes. Hsp90-inhibited cells, such as proteasome-inhibited cells, are poor stimulators of T lymphocytes. The role of hsp90 in presentation of an ovalbumin epitope is shown to be at a postproteasomal step: hsp90 associates with N-terminally extended precursors of the SIINFEHL epitope, and such peptides are depleted from hsp90 preparations in hsp90-inhibited cells. Inhibition of hsp90 in the antigen donor cell compromises their ability to cross-prime. Conversely, stressed cells expressing elevated hsp90 levels show a heat-shock factor-dependent, enhanced ability to cross-prime. These results demonstrate a substantial role for hsp90 in chaperoning of antigenic peptides in direct and indirect presentation. The introduction of a stress-inducible component in these pathways has significant implications for their modulation during fever and infection.
Stochasticity in fusion plasmas
Authors: Finken K.H.
Published: January 2008

In recent years the importance of externally imposed resonant magnetic fields on plasmas
has become more and more recognized. These fields act either at well defined plasma layers, e.g. by
ergodization or more globally by exciting large-size modes in the plasma. A major
achievement of research in recent years is the possibility to suppress large type 1 ELMS by
resonant perturbation fields which modify the transport at the plasma edge. This option has
been studied extensively at DIII-D and recently progress has also been made at JET and
TEXTOR. Several devices are preparing further experiments to study the important aim of avoiding
transient heat pulses and thus to substantially increase the lifetime of ITER. Other topics of
interest for fusion reactors are the option of distributing the heat to a large area, optimizing
methods for heat and particle exhaust, and understanding the transport around tearing
mode instabilities.

Since October 2003, three SFP workshops have taken place. Important papers from the first two
workshops have been published in previous special issues of Nuclear Fusion (stacks.iop.org/NF/44/i=6 and stacks.iop.org/NF/46/i=4)
and the contents of this issue are based on presentations to the Third Workshop on
Stochasticity in Fusion Plasmas (SFP) held in Jülich, Germany, 5–7 March 2007.

The SFP workshops have been stimulated by the installation of the Dynamic Ergodic Divertor
(DED) in the TEXTOR tokamak. It has attracted colleagues working on various plasma
configurations such as tokamaks, stellarators or reversed field pinches. The workshop is
devoted to phenomena on the plasma edge and to transport questions over the whole plasma
cross section. It is a meeting place for experimental and theoretical working groups. The
next workshop is planned for February/March 2009 in Jülich. For details see www.fzjuelich.de/sfp/.

We hope that the article series in Nuclear Fusion will stimulate interest in the fascinating
subject of plasma physics.
Numerical study of the resonant magnetic perturbations for Type I edge localized modes control in ITER
Authors: Bécoulet M. , Nardon E. , Huysmans G. , Zwingmann W. , Thomas P. , Lipa M. , Moyer R. , Evans T. , Chuyanov V. , Gribov Y. , Polevoi A. , Vayakis G. , Federici G. , Saibene G. , Portone A. , Loarte A. , Doebert C. , Gimblett C. , Hastie J. , Parail V.
Published: January 2008

A number of possible designs of external and in-vessel coils generating resonant magnetic perturbations (RMPs) for Type I edge localized modes (ELMs) control in ITER are analysed for the reference scenarios (H-mode, Hybrid and Steady-State) taking into account physical, technical and spatial constraints. The level of stochasticity (Chirikov parameter ∼1 at ψ1/2∼ 0.95) generated by the I-coils in the DIII-D experiments on ELMs suppression was taken as a reference. Designs with a toroidal symmetryn= 3 were considered to avoid lowernnumbers producing larger central islands, a potential trigger of MHD instabilities. The evaluation of RMP coils designs is done with respect to the RMPs spectrum that should produce enough edge ergodisation and minimum central perturbations at minimum current. The proposed designs include in-vessel, mid-ports and external coils. Changes in the equilibrium due to changes in the internal inductanceli, the poloidal beta βpand the edge magnetic shear in a reasonable range for ITER scenarios were demonstrated to have a small effect on the edge ergodisation. Present estimations were done without margins and for vacuum fields neglecting plasma response on RMPs. The validity of the vacuum approach was estimated analytically in thevisco-resistive linear responseregime using [1]. The typical radial magnetic field amplitudes produced by RMP coils in DIII-D and ITER are an order of magnitude or slightly above the critical values for the ‘downward’ bifurcation to the reconnected stage indicating the possibility of the islands formation in the pedestal region. Central islands (from the top of the pedestal) are expected to be screened.
Benchmarking of codes for electron cyclotron heating and electron cyclotron current drive under ITER conditions
Authors: Prater R. , Farina D. , Gribov Yu. , Harvey R.W. , Ram A.K. , Lin-Liu Y.-R. , Poli E. , Smirnov A.P. , Volpe F. , Westerhof E. , Zvonkov A.
Published: January 2008

Optimal design and use of electron cyclotron heating requires that accurate and relatively quick computer codes be available for prediction of wave coupling, propagation, damping and current drive at realistic levels of EC power. To this end, a number of codes have been developed in laboratories worldwide. A detailed comparison of these codes is desirable since they use a variety of methods for modelling the behaviour and effects of the waves. The approach used in this benchmarking study is to apply these codes to a small number of representative cases. Following minor remedial work on some codes, the agreement between codes for off-axis application is excellent. The largest systematic differences are found between codes with weakly relativistic and fully relativistic evaluation of the resonance condition, but even there the differences amount to less than 0.02 in normalized minor radius. For some other cases, for example for central current drive, the code results may differ significantly due to differences in the physics models used.
Effect of toroidal field ripple on plasma rotation in JET
Authors: de Vries P.C. , Salmi A. , Parail V. , Giroud C. , Andrew Y. , Biewer T.M. , Crombé K. , Jenkins I. , Johnson T. , Kiptily V. , Loarte A. , Lönnroth J. , Meigs A. , Oyama N. , Sartori R. , Saibene G. , Urano H. , Zastrow K.-D.
Published: January 2008

Dedicated experiments on TF ripple effects on the performance of tokamak plasmas have been carried out at JET. The TF ripple was found to have a profound effect on the plasma rotation. The central Mach number,M, defined as the ratio of the rotation velocity and the thermal velocity, was found to drop as a function of TF ripple amplitude (δ) from an average value ofM= 0.40–0.55 for operations at the standard JET ripple of δ = 0.08% toM= 0.25–0.40 for δ = 0.5% andM= 0.1–0.3 for δ = 1%. TF ripple effects should be considered when estimating the plasma rotation in ITER. With standard co-current injection of neutral beam injection (NBI), plasmas were found to rotate in the co-current direction. However, for higher TF ripple amplitudes (δ ∼ 1%) an area of counter rotation developed at the edge of the plasma, while the core kept its co-rotation. The edge counter rotation was found to depend, besides on the TF ripple amplitude, on the edge temperature. The observed reduction of toroidal plasma rotation with increasing TF ripple could partly be explained by TF ripple induced losses of energetic ions, injected by NBI. However, the calculated torque due to these losses was insufficient to explain the observed counter rotation and its scaling with edge parameters. It is suggested that additional TF ripple induced losses of thermal ions contribute to this effect.
Synthesis and Characterization of ZnO Ellipsoid-like Nanostructures
Authors: Duan Jin-xia , Wang Hao , Huang Xin-tang
Published: January 2008

ZnO ellipsoid-like structures assembled by ZnO nanrods were fabricated from common ZnO whiskers by autoclave tests and pyrolysis integrated method. X-ray diffraction, scanning electron microscopy, transmission electron microscopy, selected area electron diffraction, and high resolution transmission electron microscopy studies reveal ZnO ellipsoidal structures are single-crystals and formed from direct "oriented attachment" of nanorods. Raman and room temperature photoluminescence spectra are also discussed.
Mechanical Properties of Machined Nanostructures
Authors: Liang Ying-chun , Chen Jia-xuan , Chen Ming-jun , Tang Yu-lan , Bai Qing-shun
Published: January 2008

Three-dimensional molecular dynamics simulations have been carried out to predict the mechanical properties of a single crystalline copper with different scratching depths and no defects by embedded-atom method potential respectively. The mechanical properties for nanostructure with no defects and machined groove are investigated by various strain rates, scratched depths and scratching directions. Through the visualization technique of atomic coordination number, the onset and movement of defects in workpiece such as dislocations are analyzed under tensile loads. Work-harden formation, recrystallization behavior and the properties of rupturing process of nanostructure are exhibited at the atomic view. The relation between stress and the onset and evolvement of defects in specimen is analyzed for fundamental understanding the mechanical properties of nanostructure.
Dielectric and Pyroelectric Properties of Compositionally Graded Pb(Zr1-xTix)O3 Thin Films Prepared by Sol-gel Process
Authors: Tang Xin-gui , Jiang Li-li , Kuang Shu-juan , Ding Ai-li , L. W. Chan H.
Published: January 2008

Compositionally graded ferroelectric lead zirconate titanate Pb(Zr1-xTix)O3 (PZT) thin films were grown on Pt/Ti/SiO2/Si substrates by using a sol-gel process. The final structure consists of six layers, up-graded graded films starting from PbZrO3 on the Pt electrode to the top PZT(50) layer, it consists of no Ti, 10%Ti, 20%Ti, 30%Ti, 40%Ti, and 50%Ti respectively. Whereas films with opposite gradient are called down-graded graded films. Structure and dielectric properties of the graded films was investigated by X-ray diffraction, Auger electron spectroscopy and by impedance analysis. The up-graded and down-graded PZT films annealed at 600C, exhibited the remanent polarization values of 18.0 and 24.2 C/cm2, respectively. The typical small signal dielectric constants and loss tan at a frequency of 100 Hz were 523 and 0.018, 544, and 0.020, respectively, for up-graded and down-graded PZT thin films. The temperature dependence of pyroelectric coefficients of the graded PZT films was measured by a dynamic technique. From 20C to 82C, the pyroelectric coefficients of the up-graded and down-graded PZT films up to 374 and 407 C/m2K, respectively.
Supersaturation Control Growth of Nanoparticle ZnO and Size Distribution Control
Authors: Fu Min , Hu Ze-shan , Tang Min , Wei Xiao-ping , Shao Min-hao , Li Lan-hua , Deng Yu-lin
Published: January 2008

Nanoparticle ZnO was synthesized in non-aqueous medium. UV adsorption spectra were measured and effective mass model was used to calculate particle size in situ. A technique method named as supersaturation control growth was developed, which dealt with addition of nanoparticle suspension with small size to another suspension with big size. As a result, those small particles completely dissolved and those big ones totally grew because of dissolution degree difference between small particles and the big ones. The particle number of big particle suspension kept being a constant and the growth rate was much higher than Ostwald ripening. Main characteristic of this technique is that size distribution of nanoparticles can be narrowed provided original size difference of two suspension is big enough and original size distribution is not too broad.
Template Synthesis and Magnetic Behavior of FeNi Alloy Nanotube Arrays
Authors: Zhou Dong , Cai Li-heng , Wen Fu-shen , Li Fa-shen
Published: January 2008

FeNi nanotubes were successfully prepared in pores of anodic aluminium oxide templates by the wetting template method. By varying the deposition conditions and the parameters of the templates, the lengths and the outer as well as the inner diameters of the tubes can be tailored. The FeNi nanotubes and their arrays were characterized by transmission and scanning electron microscopy. Macroscopic magnetic measurements show the FeNi nanotube arrays to have obvious anisotropy, and the easy axis is parallel to the nanotube axis. The magnetic moment distributions in the tube walls and the magnetization reversal mechanism are discussed. Magnetic moments of the FeNi nanotube preferentially lie in the nanotube wall, but the distribution is spatially isotropic. These magnetic behaviours are due to the unique shape of the nanotube.
Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508
Authors: Morito Daisuke , Hirao Kazuyoshi , Oda Yukako , Hosokawa Nobuko , Tokunaga Fuminori , Cyr Douglas M. , Tanaka Keiji , Iwai Kazuhiro , Nagata Kazuhiro
Published: January 2008

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.
Fluid mechanics of nodal flow due to embryonic primary cilia
Authors: Smith D.J , Blake J.R , Gaffney E.A
Published: January 2008

Breaking of left–right symmetry is crucial in vertebrate development. The role of cilia-driven flow has been the subject of many recent publications, but the underlying mechanisms remain controversial. At approximately 8 days post-fertilization, after the establishment of the dorsal–ventral and anterior–posterior axes, a depressed structure is found on the ventral side of mouse embryos, termed the ventral node. Within the node, ‘whirling’ primary cilia, tilted towards the posterior, drive a flow implicated in the initial left–right signalling asymmetry. However, the underlying fluid mechanics have not been fully and correctly explained until recently and accurate characterization is required in determining how the flow triggers the downstream signalling cascades. Using the approximation of resistive force theory, we show how the flow is produced and calculate the optimal configuration to cause maximum flow, showing excellent agreement within vitromeasurements and numerical simulation, and paralleling recent analogue experiments. By calculating numerical solutions of the slender body theory equations, we present time-dependent physically based fluid dynamics simulations of particle pathlines in flows generated by large arrays of beating cilia, showing the far-field radial streamlines predicted by the theory.
Sliding-induced adhesion of stiff polymer microfibre arrays. I. Macroscale behaviour
Authors: Lee Jongho , Majidi Carmel , Schubert Bryan , Fearing Ronald S
Published: January 2008

Gecko-inspired microfibre arrays with 42 million polypropylene fibres cm
−2
(each fibre with elastic modulus 1 GPa, length 20 μm and diameter 0.6 μm) were fabricated and tested under pure shear loading conditions, after removing a preload of less than 0.1 N cm
−2
. After sliding to engage fibres, 2 cm
2
patches developed up to 4 N of shear force with an estimated contact region of 0.44 cm
2
. The control unfibrillated surface had no measurable shear force. For comparison, a natural setal patch tested under the same conditions on smooth glass showed approximately seven times greater shear per unit estimated contact region. Similar to gecko fibre arrays, the synthetic patch maintains contact and increases shear force with sliding. The high shear force observed (approx. 210 nN per fibre) suggests that fibres are in side contact, providing a larger true contact area than would be obtained by tip contact. Shear force increased over the course of repeated tests for synthetic patches, suggesting deformation of fibres into more favourable conformations.
Pedigrees, assortative mating and speciation in Darwin's finches
Authors: Grant Peter R , Grant B. Rosemary
Published: January 2008

Pedigree analysis is a useful tool in the study of speciation. It can reveal trans-generational influences on the choice of mates. We examined mating patterns in a population of Darwin's medium ground finches (Geospiza fortis) on Daphne Major Island to improve our understanding of how a barrier to the exchange of genes between populations arises in evolution. Body sizes of mates were weakly correlated. In one year, the smallest females were paired non-randomly with the males of similar size, and in another year the largest males were paired with the largest females. An influence of parental morphology on the choice of mates, as expected from sexual imprinting theory, was found; the body size of mates was predicted by the body sizes of both parents, and especially strongly by the father's. These associations imply that the seeds of reproductive isolation between species are present within a single variable population. The implication was subject to a natural test: two exceptionally large birds of the study species, apparently immigrants, bred with each other, as did their offspring, and not with the members of the resident population. The intense inbreeding represents incipient speciation. It parallels a similar phenomenon when another species, the large ground finch, immigrated to Daphne and established a new population without interbreeding with the resident medium ground finches.