bluuebunny/crossref_metadata_2025 huggingface dataset:
51 PHENOTYPIC VARIATION IN CLONED SWINE IS CORRECTED IN THE F1 GENERATION
Authors: Mir B. , Zaunbrecher G. , Piedrahita J.A.
Published: December 2004

Systematic studies of cloned animals generated from adult somatic cell nuclei are critical in assessing the utility of somatic cell cloning in various applications, including the safety of food products from cloned animals and their offspring. Studies in mice show that abnormalities seen in the cloned parents are not transmitted to the siblings. To our knowledge, however, there are no studies on the F1 progeny of clones from food animals. Previously, we compared somatic cell derived cloned pigs with naturally bred control pigs on a series of physiological and genetic parameters. Phenotypic and genetic analyses indicated that there are two classes of traits, one in which the cloned pigs have less variation than controls and another characterized by variation that is equally high in cloned and control pigs. We have extended our studies to the F1 progeny of these clones to see whether these phenotypic differences are transmitted to the next generation. Age-, sex-, and breed-matched cloned and control pigs, housed together since weaning, were used in this study. Starting with their second estrus cycle, standing gilts were mated two consecutive days. All gilts were mated to the same boar. Pregnant cloned (n = 9) and naturally bred (n = 5) gilts (F0) were allowed to farrow naturally, and number and sex of live offspring at birth (F1) recorded. There was no difference in the average litter size between litters from cloned gilts and naturally bred controls (7.78 ± 2.6 and 7.40 ± 3.0, respectively; mean ± SD) or in the degree of litter size variation (coefficients of variation of 33.4% and 40.5% for litters of clones and controls, respectively). Similarly there were no statistical differences between sex ratios from cloned litters (51%:49%; M:F) and control litters (59%:41%; M:F). Blood profiles among cloned pigs, control pigs, and their progeny were compared at two time points, i.e. 15 and 27 weeks, to quantify the effect of cloning on various blood parameters and their transmission to next generation. Although the range of values for all traits overlapped between different classes, the variation differed between F0 clones and F0 controls. In the clones there were two groups of traits: one in which cloned pigs had less variability than controls, and the other in which clones had the same variability as control pigs. In contrast, the variability between all of the traits in F1 progeny of both the clones and the control pigs was similar at 15 and 27 weeks, with one exception. Combined, our data and previous results in mice strongly support the hypothesis that offspring of clones are to all intent and purposes indistinguishable from offspring of naturally bred animals, and as such there should not be any increased risks associated with consumption of products from these animals.
This work was supported from NIH grant HL 51587 to JAP.
58 HANDMADE CLONING IN TRANS-SPECIES NT: CULTURE MEDIUM HAS AN EFFECT ON THE ABILITY OF RECONSTRUCTED BOVINE-MURINE EMBRYOS TO DEVELOP BEYOND THE 8-CELL STAGE
Authors: Nieminen P.-M. , Aho M. , Kananen-Anttila K. , Reinikainen E. , Halmekytö M.
Published: December 2004

The objectives of studies of trans-species nuclear transfer (NT) include epigenetic reprogramming and stem cell technology. The present study evaluated the effect of culture media on the development of reconstructed bovine-murine embryos. Bovine NT served as a technical control. The NT embryos were produced with the hand made cloning (HMC) technique (Vajta G et al. 2003 Biol. Reprod. 68, 571–578), and to our knowledge, this is the first report on the application of HMC in trans-species NT. Abattoir-derived bovine oocytes were matured for 21 h and enucleated by hand as described (Vajta G et al. 2003). Bovine cytoplasts were fused with either bovine granulosa cells or murine fetal fibroblasts. The bovine NT embryos were cultured for 7 days in modified SOFaaci (Holm P et al. 1999 Theriogenology 52, 683–700) containing either 5% FBS (8 trials) or 4 mg mL−1 fatty acid free albumin (FAFBSA, 6 trials). Bovine-murine NT embryos were cultured for 4.5 days in SOFaaci + FAFBSA (5 trials), or for the first 12 h in SOFaaci + FAFBSA and until 4.5 days in KSOMaa (Biggers JD 1991 J. Reprod. Fertil. 91, 543) containing 1 mg mL−1 embryo-tested BSA (4 trials). The results are shown in Table 1. In bovine NT embryos, both cleavage (day 2) and day 7 blastocyst rates were significantly improved was SOFaaci + FAFBSA was used as culture medium. Culture medium did not affect the cleavage rate of bovine-murine NT embryos at 12–16 h after start of culture. The development of reconstructed bovine-murine embryos beyond the 8-cell stage was significantly improved when SOFaaci + FAFBSA was replaced with KSOMaa + BSA after 12 h culture. Fourteen of a total of 464 (3.0%) Day 4.5 bovine-murine reconstructed embryos reached early morula stage with signs of compaction. The study showed that the development of the reconstructed NT embryos was significantly affected by the culture medium. Contrary to earlier findings (Park SH et al. 2004 Mol. Reprod. Dev. 68, 25–34), the bovine-murine reconstructed embryos developed beyond 8-cell stage, even until early compaction. The gene expression of species-specific and development-related genes of the reconstructed embryos is under characterization.

Table 1.
Effect of culture medium on cleavage and development of reconstructed NTt embryos

Professor Gabor Vajta is greatly acknowledged for his contribution in establishing the HMC technique in our laboratory.
6 VITRIFICATION OF BOVINE EMBRYOS WITHOUT ANIMAL-DERIVED PRODUCTS
Authors: Walker D. , Seidel G.
Published: December 2004

Media free of animal products would facilitate import and export requirements for embryos. Our goal was to test replacement of bovine serum albumin (BSA) (2.5 μg/mL) with sodium hyaluronate (SH) (4.2 μg/mL, 12.5 μg/mL, or 37.5 μg/mL) as the macromolecule in a very simple system in which embryos were vitrified in 0.25-mL straws suitable for direct embryo transfer. Day 7 blastocysts (n = 384) were produced in vitro with semen of 3 bulls, 2 replicates each. For vitrification, embryos were placed into chemically defined, HEPES-buffered medium (HCDM-2) and then transferred to V1 (5 M ethylene glycol, 0.5 M galactose in HCDM-2 plus BSA or SH) for 3 min. Next, embryos were placed in a 6-μL drop of V2 (7 M ethylene glycol, 0.5 M galactose, and 18% w/v Ficoll 70 in HCDM-2 plus BSA or SH) for 45 s. During equilibration, dilution medium (0.5 M galactose in HCDM-2 plus BSA or SH) was aspirated into 0.25 mL straws, followed by the 6-μL drop of V2 plus embryos, and a final short column of dilution medium. When 45 s had elapsed, the heat-sealed end of straw was dipped into liquid nitrogen to cover the embryo and then plunged slowly. Straws were thawed in air for 10 s and then in 37°C water for 20 s. Straws were then shaken like a clinical thermometer 4 times to mix columns, and held in 37°C water for 10 min before expelling embryos to be rinsed and cultured in CDM-2 + 5% FCS. Survival (as determined by expansion of blastocysts), quality score (1 = excellent, 2 = fair, 3 = poor), inner cell mass quality (ICM) (1 = large and compact, 2 = clearly visible, 3 = not discernable), and blastocyst stage (5 = early, 6 = full, 7 = expanded, 8 = hatching, 9 = hatched) were evaluated 24 h post-thaw and analyzed by ANOVA. Due to poor embryo recovery from straws in replicates 1–3, 0.1% polyvinyl alcohol (PVA) was added to vitrification solutions in replicates 4–6. Survival was calculated as a percentage of noncryopreserved controls in the same replicate (control: survival 93%, quality 1.52, ICM 1.43, stage 7.47). The lowest dose of SH replaced BSA efficaciously with this vitrification procedure (Table 1). Addition of PVA greatly improved all responses (main effects): recovery 99% vs. 69%; survival 78% vs. 53%; quality 2.07 vs. 2.43; ICM 2.10 vs. 2.47; stage 5.98 vs. 4.94; all P < 0.001. Successful vitrification of embryos in solutions containing 4.2 μg/mL SH plus PVA in place of BSA in 0.25 mL straws, from which embryos can be transferred directly, further increases the appeal of vitrification as an alternative to conventional cryopreservation.
66 IN VITRO DEVELOPMENT OF YAK (POEPHAGUS MUTUS) CLONED EMBRYOS BY INTERSPECIES SOMATIC NUCLEAR TRANSFER
Authors: Su L. , Du F.L. , Sung L.Y. , Yang S. , Jeong B.S. , Casinghino S. , Nedambale T.L. , Xu J. , Tian C.X. , Yang X. , Ji W.
Published: December 2004

Interspecies nuclear transfer (NT) is an important tool for preservation of endangered animal species. This study was carried out to clone Yak (Poephagus mutus) embryos by using Yak skin fibroblasts and bovine (Bos taurus) recipient cytoplasts, and to compare the efficiency of YAK interspecies NT (bovine cytoplast-Yak donor cell) and bovine somatic NT (bovine cytoplast-bovine donor cell). Recipient oocytes were extracted from antral follicles of bovine ovaries, and subsequently cultured in maturation medium for 18–20 h in 5% CO2 and 95% humidified air at 39°C. Cumulus cells were removed from the oocytes by vortexing also facilitated further enucleation. Yak skin fibroblast cells were prepared from cultured ear explants of an adult 5-year-old female. Fibroblasts were cultured at passage 6–9 in 10% FBS DMEM at 37°C in 5% CO2 humidified air. The donor cell at a diameter of 19–20 μm was inserted into the perivitelline space of an enucleated oocyte. A bovine female cell line at similar passage number was used for bovine somatic NT as control. Somatic cell-cytoplast pairs were then fused by applying two direct current pulses at 2.0 kV/cm for a duration of 6–10 μs/pulse. Fused embryos were activated in 10 μg/mL cycloheximide and 2.5 μg/mL cytochalasin D in M199 plus 7.5% FBS for 5 h. Reconstructed Yak embryos were cultured in CR1aa plus 6 mg/mL BSA for 2 days (initiation of activation = Day 0) at 39°C, 5% CO2, 5% O2, and 90% N2, and then in 7.5% FBS CR1aa medium for 5 successive days on bovine cumulus monolayers. Expanding and hatching blastocysts on Day 7 were recorded and cryopreserved for further embryo transfer trials. The percentage of cleavage and the development to morulae and blastocysts were statistically analyzed using a General Linear Model (GLM, Univariate, SPSS 9.0, SPSS Inc, Chicago, IL, USA). As indicated in Table 1, the results demonstrated that the efficiencies of fusion rate as well as developmental potential in vitro were significantly higher in the bovine somatic NT group compared to those of the Yak interspecies NT group. However, the morphology and cell number per embryo of interspecies Yak cloned embryos were indistinguishable from those of bovine NT embryos. Our data suggest that bovine oocytes possess the capability of reprogramming/reactivation of the genome from differentiated somatic Yak nuclei.

Table 1.
Comparison of yak interspecies and bovine somatic nuclear transfer
Muscle ring finger protein-1 inhibits PKCε activation and prevents cardiomyocyte hypertrophy
Authors: Arya Ranjana , Kedar Vishram , Hwang Jae Ryoung , McDonough Holly , Li Hui-Hua , Taylor Joan , Patterson Cam
Published: December 2004

Much effort has focused on characterizing the signal transduction cascades that are associated with cardiac hypertrophy. In spite of this, we still know little about the mechanisms that inhibit hypertrophic growth. We define a novel anti-hypertrophic signaling pathway regulated by muscle ring finger protein-1 (MURF1) that inhibits the agonist-stimulated PKC-mediated signaling response in neonatal rat ventricular myocytes. MURF1 interacts with receptor for activated protein kinase C (RACK1) and colocalizes with RACK1 after activation with phenylephrine or PMA. Coincident with this agonist-stimulated interaction, MURF1 blocks PKCε translocation to focal adhesions, which is a critical event in the hypertrophic signaling cascade. MURF1 inhibits focal adhesion formation, and the activity of downstream effector ERK1/2 is also inhibited in the presence of MURF1. MURF1 inhibits phenylephrine-induced (but not IGF-1–induced) increases in cell size. These findings establish that MURF1 is a key regulator of the PKC-dependent hypertrophic response and can blunt cardiomyocyte hypertrophy, which may have important implications in the pathophysiology of clinical cardiac hypertrophy.
BDNF-induced recruitment of TrkB receptor into neuronal lipid rafts
Authors: Suzuki Shingo , Numakawa Tadahiro , Shimazu Kazuhiro , Koshimizu Hisatsugu , Hara Tomoko , Hatanaka Hiroshi , Mei Lin , Lu Bai , Kojima Masami
Published: December 2004

Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity but the underlying signaling mechanisms remain unknown. Here, we show that BDNF rapidly recruits full-length TrkB (TrkB-FL) receptor into cholesterol-rich lipid rafts from nonraft regions of neuronal plasma membranes. Translocation of TrkB-FL was blocked by Trk inhibitors, suggesting a role of TrkB tyrosine kinase in the translocation. Disruption of lipid rafts by depleting cholesterol from cell surface blocked the ligand-induced translocation. Moreover, disruption of lipid rafts prevented potentiating effects of BDNF on transmitter release in cultured neurons and synaptic response to tetanus in hippocampal slices. In contrast, lipid rafts are not required for BDNF regulation of neuronal survival. Thus, ligand-induced TrkB translocation into lipid rafts may represent a signaling mechanism selective for synaptic modulation by BDNF in the central nervous system.
Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in β-cells
Authors: Trajkovski Mirko , Mziaut Hassan , Altkrüger Anke , Ouwendijk Joke , Knoch Klaus-Peter , Müller Stefan , Solimena Michele
Published: December 2004

Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic β-cells. Here, we show that exocytosis of SGs and insertion of ICA512 in the plasma membrane promotes the Ca2+-dependent cleavage of ICA512 cytoplasmic domain by μ-calpain. This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression. Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in β-cells, whose impaired insulin production and secretion causes diabetes.
Targeted deletion of numb and numblike in sensory neurons reveals their essential functions in axon arborization
Authors: Huang Eric J. , Li Huashun , Tang Amy A. , Wiggins Amanda K. , Neve Rachael L. , Zhong Weimin , Jan Lily Y. , Jan Yuh Nung
Published: December 2004

Mouse Numb homologs antagonize Notch1 signaling pathways through largely unknown mechanisms. Here we demonstrate that conditional mouse mutants with deletion of numb and numblike in developing sensory ganglia show a severe reduction in axonal arborization in afferent fibers, but no deficit in neurogenesis. Consistent with these results, expression of Cre recombinase in sensory neurons from numb conditional mutants results in reduced endocytosis, a significant increase in nuclear Notch1, and severe reductions in axon branch points and total axon length. Conversely, overexpression of Numb, but not mutant Numb lacking α-adaptin-interacting domain, leads to accumulation of Notch1 in markedly enlarged endocytic–lysosomal vesicles, reduced nuclear Notch1, and dramatic increases in axonal length and branch points. Taken together, our data provide evidence for previously unidentified functions of Numb and Numblike in sensory axon arborization by regulating Notch1 via the endocytic–lysosomal pathways.
Machine vision in the electronics industry
Authors: Plain‐Jones Charlie , Ludlow Jonathan E
Published: December 2004

Focuses on the utilization of machine vision in three areas of
electronic assembly. Looks at odd‐shaped component placement covering
part location, target location and placement. Discusses LCD display
inspection, including LCD, LED and Plasma technologies. Also examines machine
vision in automated die and wire bond inspection for multi‐chip module
manufacturing. Considers the benefits of automating inspection processes and
concludes by looking at how useful measurements can be extracted from the
analysis of images of wire bonds.
Justifying flexible automation for PCB assembly
Authors: Holcomb Gregory W
Published: December 2004

Examines the use of robots for printed circuit board assembly with
particular reference to the growing trend of mixing surface‐mount and
through‐hole technology on to a single board. Describes the
flexibility of today’s robotic cells, with one system capable of
assembling a range of PCBs, making changes to board assembly by changing the
software instead of the hardware. Looks at the various end effectors for
robotic assembly of PCBs, including a new type of gripper which can handle
all variations of circuit boards without having to make tooling or gripper
changes.
Communities, Opportunities, and Adolescents’ Sexual Behavior in KwaZulu‐Natal, South Africa
Authors: Kaufman Carol E. , Clark Shelley , Manzini Ntsiki , May Julian
Published: December 2004

In 2002, the prevalence of HIV among South African clinic attendees under the age of 20 was 15 percent, suggesting a correspondingly high level of unprotected sex and risky sexual behavior. Past research focused on the dynamics of individuals’ and partners’ sexual decisionmaking without accounting for the larger context in which such decisions are made. Do adolescents’ opportunities for schooling, work, and other community activities make a difference? This study uses data collected from a representative sample of young people living in KwaZulu‐Natal, South Africa, to explore these questions. Logit analyses show that for girls, higher levels of education reduced the probability of having had sex in the 12 months prior to the survey, and average wages were positively associated with condom use. Greater participation in community sports increased risk‐taking behaviors among boys but decreased them among girls. Within the household, education of adults had a positive effect on condom use for both boys and girls.
The beneficial effects of treatment with all-trans-retinoic acid plus corticosteroid on autoimmune nephritis in NZB/WF1 mice
Authors: Nozaki Y , Yamagata T , Yoo B-S , Sugiyama M , Ikoma S , Kinoshita K , Funauchi M , Kanamaru A
Published: December 2004

Corticosteroids are highly effective anti-inflammatory or immunosuppressive drugs used commonly to treat human systemic lupus erythematosus (SLE). All-trans-retinoic acid (ATRA), which belongs to a class of retinoids that exert immunomodulatory and anti-inflammatory functions, can also suppress the development of lupus nephritis in an animal model. However, both agents can inflict serious adverse effects. Here, we have asked whether ATRA can serve as a steroid-sparing drug in the treatment of lupus nephritis. To examine the efficacy of combining predonisolone (PSL) with ATRA, we treated intraperitoneally New Zealand black/white F1 (NZB/W F1) mice with PSL, ATRA or both agents. Survival rate and proteinuria were determined once a month. Cytokine and anti-DNA antibody production were determined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Renal histopathology was observed by haematoxylin and periodic acid Schiff (PAS), immunoperoxidase and immunohistochemical assay. Survival rate and proteinuria were improved in all experimental groups, and were much improved in the mice receiving the combination of ATRA and PSL (P < 0·05). A single administration of ATRA reduced the Th1 [interleukin (IL)-2, interferon (IFN)-γ and IL-12], and a Th2 (IL-4) cytokine level, as effectively as administration of PSL. ATRA also suppressed the expression of inducible nitric oxide synthetase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) in the kidney. The combination of PSL and ATRA significantly reduced IgG2 (especially IgG2b)-specific anti-DNA antibody levels in comparison with administration of either agent alone. These data suggest that ATRA might have the potential to act as a new therapeutic and steroid-sparing drug against lupus nephritis.
Influence of age and duration of infection on bacterial load and immune responses to Helicobacter pylori infection in a murine model
Authors: Minoura T , Kato S , Otsu S , Kodama M , Fujioka T , Iinuma K , Nishizono A
Published: December 2004

Using a murine model, we previously showed that Helicobacter pylori infects and colonizes offspring via maternal transmission during the nursing period. The aim of this study was to investigate the influence of age and duration of infection on inflammatory and immune responses to H. pylori in infant and adult mice. During the breast-feeding period, the number of bacteria was significantly suppressed in 1-week-old mice infected with H. pylori at an early stage of nursing, compared with adult mice, suggesting that breast-milk induces such low colonization. In addition, these mice had weaker gastric inflammation, especially Th1 cytokine and humoral responses than in mice infected with H. pylori after weaning in spite of elevated levels of Th1 cytokines. Although infant mice showed low inflammatory responses against H. pylori, they produced H. pylori-specific antibodies following vaccination with oral or parenteral adjuvant. Our results suggest the importance of age at the time of primary infection on bacterial load, gastric inflammation and humoral responses in a murine model of H. pylori infection.
Endogenous glucocorticoids modulate neutrophil function in a murine model of haemolytic uraemic syndrome
Authors: Gómez S A , Fernández G C , Camerano G , Dran G , Rosa F A , Barrionuevo P , Isturiz M A , Palermo M S
Published: December 2004

Haemolytic uraemic syndrome (HUS) is caused by Shiga-toxin-producing Escherichia coli (STEC). Although, Shiga toxin type 2 (Stx2) is responsible for the renal pathogenesis observed in patients, the inflammatory response, including cytokines and polymorphonuclear neutrophils (PMN), plays a key role in the development of HUS. Previously, we demonstrated that Stx2 injection generates an anti-inflammatory reaction characterized by endogenous glucocorticoid (GC) secretion, which attenuates HUS severity in mice. Here, we analysed the effects of Stx2 on the pathogenic function of PMN and the potential role of endogenous GC to limit PMN activation during HUS development in a murine model. For this purpose we assessed the functional activity of isolated PMN after in vivo treatment with Stx2 alone or in simultaneous treatment with Ru486 (GC receptor antagonist). We found that Stx2 increased the generation of reactive oxygen intermediates (ROI) under phobol-myristate-acetate (PMA) stimulation and that the simultaneous treatment with Ru486 strengthened this effect. Conversely, both treatments significantly inhibited in vitro phagocytosis. Furthermore, Stx2 augmented in vitro PMN adhesion to fibrinogen (FGN) and bovine serum albumin (BSA) but not to collagen type I (CTI). Stx2 + Ru486 caused enhanced adhesion to BSA and CTI compared to Stx2. Whereas Stx2 significantly increased migration towards N-formyl-methionyl-leucyl-phenylalanine (fMLP), Stx2 + Ru486 treatment enhanced and accelerated this process. The percentage of apoptotic PMN from Stx2-treated mice was higher compared with controls, but equal to Stx2 + Ru486 treated mice. We conclude that Stx2 activates PMN and that the absence of endogenous GC enhances this activation suggesting that endogenous GC can, at least partially, counteract PMN inflammatory functions.
Enhanced interleukin-18 levels in the peripheral blood of children with coeliac disease
Authors: Lettesjö H , Hansson T , Bergqvist Å , Grönlund J , Dannaeus A
Published: December 2004

Coeliac disease (CoD) is a small intestinal disorder characterized by villous atrophy, crypt cell hyperplasia and an increased production of T helper cell type 1 (Th1) cytokines. Interleukin (IL)-18 is a pro-inflammatory cytokine that has a crucial role in maintaining the Th1 response. In this study, the serum levels of IL-18 were measured in children with CoD or other gastrointestinal diseases in order to evaluate the possibility of using IL-18 as a disease activity marker. IL-18 levels were higher in samples from CoD patients [median 443 pg/ml (148–885)] compared to healthy controls [median 205 pg/ml (11–379)], P < 0·05. In contrast, the levels of IL-18 were not enhanced significantly in the serum from patients with inflammatory bowel disease (IBD) [median 324 pg/ml (207–546)] or in the disease control group [median 303 pg/ml (2–689)]. In CoD patients, after 2 weeks of gluten challenge (GC), serum IL-18 was unchanged [median 268 pg/ml (59–458)] compared to patients on a gluten-free diet [median 220 pg/ml (53–600)], while IL-18 was increased after 12 weeks of GC [median 551 pg/ml (94–952)], P < 0·01. The IL-18 levels correlated with IgA anti-transglutaminase antibody levels (rs = 0·59, P = 0·016) in serum from untreated CoD patients, and IL-18 also followed the degree of small intestinal villous atrophy in 12 out of 19 CoD patients. Our results support the view that serum IL-18 concentrations in children with CoD follow disease activity, suggesting a role for IL-18 in the induction of an inflammatory Th1-response after gluten exposure.
Contribution of Va24+Vb11+ natural killer T cells in Wilsonian hepatitis
Authors: Kinebuchi M , Matsuura A , Ohya K , Abo W , Kitazawa J
Published: December 2004

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. There is no evidence that the WD patient's immune system attacks copper accumulated hepatocytes. Here we describe that the frequency and absolute number of Vα24+Vβ11+ natural killer T (NKT) cells were significantly increased in 3 cases of WD, whereas those of CD3+CD161+ NKT cells were within the normal range. Patients no. 1 and 2 had a presymptomatic form of WD. Their tissue specimens showed pathological changes of mild degeneration of hepatocytes with a few infiltrating mononuclear cells and a low degree of fatty change. Patient no. 3 displayed fulminant hepatitis with Coombs-negative haemolytic anaemia. The tissue specimens of patient no. 3 showed macronodular cirrhosis with thick fibrosis, inflammatory infiltrates and spotty necrosis. Human Vα24+Vβ11+ NKT cells are almost equal to CD1d-restricted NKT cells. Therefore we investigated CD1d-restricted NKT cells in the LEC rat as an animal model of WD. In LEC rats before hepatitis onset, the number and phenotype of liver NKT cells were normal. At about 4 months of age all LEC rats developed acute hepatitis accompanied by acute jaundice, and CD161high NKT cells developed in their livers. CD161highαβTCRbright NKT cells developed in some of them. Their hepatitis was severe. CD161highαβTCRbright NKT cells expressed an invariant rat Vα14-Jα281 chain, which is CD1d-restricted. Furthermore, liver lymphocytes in the acute jaundiced LEC rats with CD161highαβTCRbright NKT cells had significant and CD1d-specific cytotoxic activity.
The bisphosphonate acute phase response: rapid and copious production of proinflammatory cytokines by peripheral blood gd T cells in response to aminobisphosphonates is inhibited by statins
Authors: Hewitt R E , Lissina A , Green A E , Slay E S , Price D A , Sewell A K
Published: December 2004

The bisphosphonates are a novel class of drug that have been registered for various clinical applications worldwide. Bisphosphonates, and in particular the aminobisphosphonates (nBPs), are known to have a number of side-effects including a rise in body temperature and accompanying flu-like symptoms that resemble a typical acute phase response. The mechanism for this response has been partially elucidated and appears to be associated with the release of tumour necrosis factor (TNF)α and interleukin (IL)6, although the effector cells that release these cytokines and the mechanism of action remain enigmatic. Here, we show that the nBP-induced acute phase response differs from the typical acute phase response in that CD14+ cells such as monocytes and macrophages are not the primary cytokine producing cells. We show that by inhibiting the mevalonate pathway, nBPs induce rapid and copious production of TNFα and IL6 by peripheral blood γδ T cells. Prior treatment with statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, blocks nBP-induced production of these proinflammatory cytokines by γδ T cells and may offer a means of avoiding the associated acute phase response. In addition, our findings provide a further mechanism for the anti-inflammatory effects attributed to inhibitors of HMG CoA reductase.
Intracellular cytokines in blood T cells in lung transplant patients – a more relevant indicator of immunosuppression than drug levels
Authors: Hodge G , Hodge S , Reynolds P , Holmes M
Published: December 2004

Allograft rejection remains a major cause of morbidity and mortality following lung transplantation and is associated with an increase in T-cell pro-inflammatory cytokine expression. Systemic levels of immunosuppressive drugs used to reduce pro-inflammatory cytokine expression are closely monitored to their ‘therapeutic range’. However, it is currently unknown if levels of these drugs correlate with pro-inflammatory cytokine expression in peripheral blood T cells. To investigate the immunomodulatory effects of currently used immunosuppressive regimes on peripheral blood T-cell cytokine production, whole blood from stable lung transplant patients and control volunteers  were  stimulated in vitro and cytokine production by CD8+ and CD4+ T-cell subsets determined using multiparameter flow cytometry. T-cell IL-2 and TNFα production was significantly reduced from lung transplant patients compared to controls. CD4+ T-cell production of IFNγ was also significantly reduced from lung transplant patients but production of IFNγ by CD8+ T cells remained unchanged. There was an excellent correlation between the percentage of CD8+ T cells and the percentage of CD8+ T cells producing IFNγ from transplant patients. T-cell IL-4 and CD8+ T-cell production of TGFβ was significantly increased from lung transplant patients. We now provide evidence that current immunosuppression protocols have limited effect on peripheral blood IFNγ production by CD8+ T-cells but do up-regulate T-cell anti-inflammatory cytokines. Drugs that effectively reduce IFNγ production by CD8+ T cells may improve current protocols for reducing graft rejection in these patients. Intracellular cytokine analysis using flow cytometry may be a more appropriate indicator of immunosuppression than drug levels in these patients. This technique may prove useful in optimizing therapy for individual patients.
Impaired functional capacities of liver dendritic cells from murine hepatitis B virus (HBV) carriers: relevance to low HBV-specific immune responses
Authors: Hasebe A , Akbar S M F , Furukawa S , Horiike N , Onji M
Published: December 2004

The chronic hepatitis B virus (HBV) carrier exhibits ongoing replication of HBV and expresses abundant amounts of HBV-related antigens in the liver. However, HBV-specific immune responses are either absent or narrowly focused in these subjects. With the postulation that impaired functional abilities of liver dendritic cells (DCs) might be responsible for this, we assessed the functions of liver DCs in HBV transgenic mice (HBV-TM), an animal model of the HBV carrier state. Liver DCs were isolated from normal C57BL/6 mice and HBV-TM without the use of cytokines or growth factors. Lymphoproliferative assays were conducted to evaluate the ability of liver DCs to induce the proliferation of allogenic T lymphocytes and hepatitis B surface antigen (HBsAg)-enriched T lymphocytes. Liver DCs were stimulated with viral and bacterial products to assess their cytokine-producing capacities. In comparison to liver DCs from normal C57BL/6 mice, liver DCs from HBV-TM exhibited significantly decreased T cell proliferation-inducing capacities in allogenic mixed leucocyte reaction (P < 0·05) and HBsAg-enriched T lymphocytes proliferation assays (P < 0·05). Liver DCs from HBV-TM produced significantly lower levels of interleukin-12p70, tumour necrosis factor-alpha, interferon-gamma, and interleukin-6 (P < 0·05) compared to liver DCs from normal C57BL/6 mice. This study provides evidence that liver DCs from HBV-TM had impaired ability to induce both innate and adaptive immune responses. This might account for a weak and almost undetectable HBV-specific immune response in chronic HBV carriers. This inspires hope that up-regulation of the functions of liver DCs in situ may have therapeutic implications in chronic HBV carriers.
Rust resistance in Salix to Melampsora larici‐epitea
Authors: Pei M. H. , Ruiz C. , Bayon C. , Hunter T.
Published: December 2004

A total of 174 Salix (willow) clones belonging to 57 species and 14 interspecific hybrids were inoculated with seven pathotypes of Melampsora larici‐epitea using the leaf disc method. Infection types were scored based on the uredinial pustule area data and the inoculum density. A close correlation (R2 = 0·82) was found between the average pustule area and the average number of spores produced. Most of the willows were also assessed in the field for rust in 1999. Most willow clones belonging to the species native to western Europe were infected by the rust. In inoculation experiments, uredinia developed on 46 S. viminalis clones, out of a total of 47. In the field, all the S. viminalis clones were infected by rust. Within the subgenus Vetrix, eight out of the 17 willow species that originated from North and South America produced rust pustules in inoculation experiments. Of these, S. pellita was most susceptible. Salix irrorata and S. lasiolepsis var. bracelinae produced well developed pustules after inoculation but no rust infections were detected in the field. In both leaf disc tests and field assessments, no rust infections were found on S. candida, S. cordata, S. drummondiana, S. eriocephala, S. hookeriana, S. houghtonii, S. humilis, S. rigida var. mackenziana and S. syrticola. Of 12 species of subgenus Vetrix native to northeast Asia and Japan, only S. kochiana was susceptible both in inoculation tests and in the field. Salix rossica produced no symptoms in leaf disc tests but showed low levels of infection in the field. The maximum infection type scores in leaf disc tests were highly significantly correlated with field disease severity scores (Spearman rank correlation coefficient was 0·76, P < 1 × 10−10).
Characterization of chickpea differentials for pathogenicity assay of ascochyta blight and identification of chickpea accessions resistant to Didymella rabiei
Authors: Chen W. , Coyne C. J. , Peever T. L. , J. Muehlbauer F.
Published: December 2004

Forty‐eight chickpea germplasm lines, including 22 differentials used in previous studies, were characterized for disease phenotypes following inoculation with six isolates of Didymella (anamorph Ascochyta) rabiei, representing a wide spectrum of pathogenic variation. Representative isolates were also directly compared with six previously identified races on eight chickpea genotypes. Many of the chickpea differentials reacted similarly to inoculation with each isolate of D. rabiei, and several previously identified races caused similar levels of disease on the differentials. This indicates that the number of differentials can be reduced significantly without sacrificing accuracy in describing pathogenic variation of D. rabiei on chickpea. Pathogenic variation among samples of US isolates allowed classification of the isolates into two pathotypes. The distribution of disease phenotypes of the 48 germplasm lines was bimodal after inoculation with pathotype I isolates, whereas the distribution of disease phenotypes was continuous after inoculation with pathotype II isolates. Such distinct distribution patterns suggest that chickpea plants employ different resistance mechanisms to each pathotype and that the two pathotypes may have different genetic mechanisms controlling pathogenicity. The advantages of using the two‐pathotype system in assaying pathogenicity of the pathogen and in studying resistance mechanisms of the host are discussed. Three chickpea accessions, PI 559361, PI 559363 and W6 22589, showed a high level of resistance to both pathotypes, and can be employed as resistance sources in chickpea breeding programmes for resistance to ascochyta blight.
Predicting light leaf spot (Pyrenopeziza brassicae) risk on winter oilseed rape (Brassica napus) in England and Wales, using survey, weather and crop information
Authors: Welham S. J. , Turner J. A. , Gladders P. , Fitt B. D. L. , Evans N. , Baierl A.
Published: December 2004

Data from surveys of winter oilseed rape crops in England and Wales in growing seasons with harvests in 1987–99 were used to construct statistical models to predict, in autumn (October), the incidence of light leaf spot caused byPyrenopeziza brassicaeon winter oilseed rape crops the following spring (March/April), at both regional and individual crop scales. Regions (groups of counties) with similar seasonal patterns of incidence (percentage of plants affected) of light leaf spot were defined by using principal coordinates analysis on the survey data. At the regional scale, explanatory variables for the statistical models were regional weather (mean summer temperature and mean monthly winter rainfall) and survey data for regional light leaf spot incidence (percentage of plants with affected pods) in July of the previous season. At the crop scale, further explanatory variables were crop cultivar (light leaf spot resistance rating), sowing date (number of weeks before/after 1 September), autumn fungicide use and light leaf spot incidence in autumn. Risk of severe light leaf spot (> 25% plants affected) in a crop in spring was also predicted, and uncertainty in predictions was assessed. The models were validated using data from spring surveys of winter oilseed rape crops in England and Wales from 2000 to 2003, and reasons for uncertainty in predictions for individual crops are discussed.
NARG2 encodes a novel nuclear protein with (S/T)PXX motifs that is expressed during development
Authors: Sugiura Naoaki , Dadashev Vladimir , Corriveau Roderick A.
Published: December 2004

We previously identified a partial expressed sequence tag clone corresponding to NARG2 in a screen for genes that are expressed in developing neurons and misexpressed in transgenic mice that lack functional N‐methyl‐d‐aspartate receptors. Here we report the first characterization of the mouse and human NARG2 genes, cDNAs and the proteins that they encode. Mouse and human NARG2 consist of 988 and 982 amino acids, respectively, and share 74% identity. NARG2 does not display significant homology to other known genes, and lower organisms such as Saccharomyces cerevisiae, Drosophila melanogaster and Fugu rubripes appear to lack NARG2 orthologs. In vitro translation of the mouse cDNA yields a 150 kDa protein. NARG2 localizes to the nucleus in transfected cells, and deletion of a canonical basic nuclear localization signal suggests that this and other sequences in the protein cooperate for nuclear targeting. NARG2 consists of 16 exons in both mice and humans, 11 of which are identical in length, and alternative splicing is evident in both species. Exon 10 is the largest, and exhibits a much higher rate of nonsynonymous nucleotide substitution than the others. In addition, NARG2 contains (S/T)PXX motifs (11 in mouse NARG2, six in human NARG2). Northern blot analysis and RNase protection demonstrated that NARG2 is expressed at relatively high levels in dividing and immature cells, and that it is down‐regulated upon terminal differentiation. The results indicate that NARG2 encodes a novel (S/T)PXX motif‐containing nuclear protein, and suggest that NARG2 may play an important role in the early development of a number of different cell types.
Regulation of STAT signalling by proteolytic processing
Authors: Hendry Lisa , John Susan
Published: December 2004

Interaction of cytokines with their cognate receptors leads to the activation of latent transcription factors, the signal transducer and activator of transcription (STAT) proteins. Numerous studies have identified the critical roles played by STAT proteins in regulating cell proliferation, differentiation and survival. Consequently, the activity of STAT proteins is negatively regulated by a variety of different mechanisms, which include alternative splicing, covalent modifications, protein–protein interactions with negative regulatory proteins and proteolytic processing by proteases. Cleavage of STAT proteins by proteases results in the generation of C‐terminally truncated proteins, called STATγ, which lack the transactivation domain and behave as functional dominant‐negative proteins. Currently, STATγ isoforms have been identified for Stat3, Stat5a, Stat5b and Stat6 in different cellular contexts and biological processes. Evidence is mounting for the role of as yet unidentified serine proteases in the proteolytic processing of STAT proteins, although at least one cysteine protease, calpain is also known to cleave these STATs in platelets and mast cells. Recently, studies of acute myeloid leukaemia and cutaneous T cell lymphoma patients have revealed important roles for the aberrant expression of Stat3γ and Stat5γ proteins in the pathology of these diseases. Together, these findings indicate that proteolytic processing is an important mechanism in the regulation of STAT protein biological activity and provides a fertile area for future studies.
Weapons of STAT destruction
Authors: Horvath Curt M.
Published: December 2004

The signal transducer and activator of transcription (STAT) family of proteins function to activate gene transcription downstream of myriad cytokine and growth factor signals. The prototype STAT proteins, STAT1 and STAT2, are required for innate and adaptive antimicrobial immune responses that result from interferon signal transduction. While many viruses have evolved the ability to avoid these antiviral cytokines, the Paramyxoviruses are distinct in their abilities to interfere directly with STAT proteins. Individual paramyxovirus species differ greatly in their precise mechanism of STAT signaling evasion, but a virus‐encoded protein called V plays a central role in this process. The theme of V‐dependent interferon evasion and its variations provide significant insights into virus–host interactions and viral immune evasion that can help define targets for antiviral drug design. Exposure of the viral weapons of STAT destruction may also be instructive for application to STAT‐directed therapeutics for diseases characterized by STAT hyperactivity.
Structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus
Authors: Cheng Shu‐Fang , Wu Cheng‐Wei , Kantchev Eric Assen B , Chang Ding‐Kwo
Published: December 2004

The structure and membrane interaction of the internal fusion peptide (IFP) fragment of the avian sarcoma and leucosis virus (ASLV) envelope glycoprotein was studied by an array of biophysical methods. The peptide was found to induce lipid mixing of vesicles more strongly than the fusion peptide derived from the N‐terminal fusion peptide of influenza virus (HA2‐FP). It was observed that the helical structure was enhanced in association with the model membranes, particularly in the N‐terminal portion of the peptide. According to the infrared study, the peptide inserted into the membrane in an oblique orientation, but less deeply than the influenza HA2‐FP. Analysis of NMR data in sodium dodecyl sulfate micelle suspension revealed that Pro13 of the peptide was located near the micelle–water interface. A type II β‐turn was deduced from NMR data for the peptide in aqueous medium, demonstrating a conformational flexibility of the IFP in analogy to the N‐terminal FP such as that of gp41. A loose and multimodal self‐assembly was deduced from the rhodamine fluorescence self‐quenching experiments for the peptide bound to the membrane bilayer. Oligomerization of the peptide and its variants can also be observed in the electrophoretic experiments, suggesting a property in common with other N‐terminal FP of class I fusion proteins.
Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde‐3‐phosphate dehydrogenase in the assembly process of a bi‐enzyme complex involved in CO2 assimilation
Authors: Graciet Emmanuelle , Mulliert Guillermo , Lebreton Sandrine , Gontero Brigitte
Published: December 2004

The glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) in the chloroplast of Chlamydomonas reinhardtii is part of a complex that also includes phosphoribulokinase (PRK) and CP12. We identified two residues of GAPDH involved in protein–protein interactions in this complex, by changing residues K128 and R197 into A or E. K128A/E mutants had a Km for NADH that was twice that of the wild type and a lower catalytic constant, whatever the cofactor. The kinetics of the mutant R197A were similar to those of the wild type, while the R197E mutant had a lower catalytic constant with NADPH. Only small structural changes near the mutation may have caused these differences, since circular dichroism and fluorescence spectra were similar to those of wild‐type GAPDH. Molecular modelling of the mutants led to the same conclusion. All mutants, except R197E, reconstituted the GAPDH–CP12 subcomplex. Although the dissociation constants measured by surface plasmon resonance were 10–70‐fold higher with the mutants than with wild‐type GAPDH and CP12, they remained low. For the R197E mutation, we calculated a 4 kcal/mol destabilizing effect, which may correspond to the loss of the stabilizing effect of a salt bridge for the interaction between GAPDH and CP12. All the mutant GAPDH–CP12 subcomplexes failed to interact with PRK and to form the native complex. The absence of kinetic changes of all the mutant GAPDH–CP12 subcomplexes, compared to wild‐type GAPDH–CP12, suggests that mutants do not undergo the conformation change essential for PRK binding.
Universal positions in globular proteins
Authors: Papandreou Nikolaos , Berezovsky Igor N. , Lopes Anne , Eliopoulos Elias , Chomilier Jacques
Published: December 2004

The description of globular protein structures as an ensemble of contiguous ‘closed loops’ or ‘tightened end fragments’ reveals fold elements crucial for the formation of stable structures and for navigating the very process of protein folding. These are the ends of the loops, which are spatially close to each other but are situated apart in the polypeptide chain by 25–30 residues. They also correlate with the locations of highly conserved hydrophobic residues (referred to as topohydrophobic), in a structural alignment of the members of a protein family. This study analysed these positions in 111 representatives of different protein folds, and then carried out dynamic Monte Carlo simulations of the first steps of the folding process, aimed at predicting the origins of the assembling folds. The simulations demonstrated that there is an obvious trend for certain sets of residues, named ‘mostly interacting residues’, to be buried at the early stages of the folding process. Location of these residues at the loop ends and correlation with topohydrophobic positions are demonstrated, thereby giving a route to simulations of the protein folding process.