bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of centrifugation and electrical activation on male pronucleus formation and embryonic development of porcine oocytes reconstructed with intracytoplasmic sperm injection
Authors: Yong Hwan Yul , Hong Ji Young , Pak Sun Il , Kang Sung Keun , Lee Byeong Chun , Lee Eun Song , Hwang Woo Suk
Published: May 2005

Oocyte centrifugation and electrical activation are commonly used in intracytoplasmic sperm injection (ICSI) of bovine and porcine oocytes, to facilitate visual identification of sperm release into the ooplasm and to support oocyte activation following injection with tail membrane-damaged sperm. The present study evaluated the necessity of these steps in porcine modified ICSI. In the first series of experiments, in vitro-matured gilt oocytes with or without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail first. Oocytes without centrifugation exhibited a significantly higher normal fertilisation rate, defined as male pronucleus (MPN) and female pronucleus (FPN) formation and the presence of two polar bodies, than centrifuged oocytes (40% v. 9%, respectively; P < 0.05). The rate of MPN formation was significantly higher in uncentrifuged oocytes compared with centrifuged oocytes (48% v. 17%, respectively; P < 0.05). The rates of survival, cleavage, blastocyst formation and total cell number in blastocysts did not differ between the two groups of oocytes. Next, the effect of electrical activation after ICSI on uncentrifuged oocytes injected with head membrane-damaged spermatozoa was determined. No significant differences were observed in the rate of MPN formation in sperm-injected oocytes regardless of electrical activation. However, the survival rates of sperm-injected or control oocytes without electrical activation were significantly higher than those of sperm-injected or control oocytes with electrical activation (88% and 84% v. 77% and 64%, respectively; P < 0.05). The cleavage rates of sperm-injected oocytes were significantly higher than those of control oocytes, regardless of electrical activation (77% and 81% v. 47% and 61% in sperm-injected and control oocytes with or without electrical activation, respectively; P < 0.05). Although development to blastocysts was similar in all experimental groups, the total cell numbers in blastocysts from control oocytes were significantly higher than those in sperm-injected oocytes, regardless of electrical activation (40 and 44 v. 22 and 26 in control and sperm-injected oocytes with or without electrical activation, respectively; P < 0.05). In conclusion, the present study clearly demonstrated that oocyte centrifugation before sperm injection is not beneficial to normal fertilisation and that electrical activation is not necessary in the modified porcine ICSI.
Effect of hypobaric hypoxia on lamb intrauterine growth: comparison between high- and low-altitude native ewes
Authors: Parraguez Víctor H. , Atlagich Miljenko , Díaz Rodrigo , Bruzzone María E. , Behn Claus , Raggi Luis A.
Published: May 2005

The present studies assessed the effect of hypobaric hypoxia on fetal lamb growth in high-altitude (HA) and low-altitude (LA) native ewes. Growth patterns of fetal biparietal diameter (BPD), abdominal diameter (AD) and thorax height (TH) were described by consecutive ultrasound measurements throughout the entire pregnancy. Three groups of animals were used: (1) pregnant LA ewes kept at LA (control; ‘LL’ group); (2) pregnant LA ewes moved to HA immediately after confirmation of pregnancy (‘LH’ group); and (3) pregnant HA ewes kept at HA throughout the entire pregnancy (‘HH’ group). The slope of the BPD curve was higher in LL fetuses followed by that in LH fetuses. During the last month of pregnancy, TH was higher in LH and HH fetuses, whereas AD was higher in LL than in LH fetuses. The length of gestation was longer in HH ewes (153.2 ± 4.3 days) than in LH and LL ewes (146.0 ± 5.5 and 145.0 ± 3.0 days, respectively). Bodyweight at birth was higher for LL newborns (4.2 ± 0.3 kg) than for LH and HH newborns (3.0 ± 0.5 and 3.2 ± 0.8 kg, respectively), whereas placental weight was higher in the HH group (396 ± 80 g) than in the LH (303 ± 64 g) and LL (280 ± 40 g) groups. In conclusion, an HA environment modifies fetal growth and pregnancy outcome with the magnitude of effects depending on the time of residence at HA.
Comparison of two different methods for the vitrification of hatched blastocysts from the dromedary camel (Camelus dromedarius)
Authors: Skidmore J. A. , Billah M. , Loskutoff N. M.
Published: May 2005

The uteri of 32 donor camels were flushed non-surgically on Day 6, 7 or 8 after ovulation and a total of 184 embryos was recovered. Sixty Day 6 embryos and 61 Day 7 embryos were vitrified or frozen ultrarapidly using open pulled straws and a modified version of the Vajta protocol. These embryos were subjected to concentrations of either 10% and 20% or 20% and 40% ethanediol as the cryoprotectant before being loaded into open pulled straws (OPS) and plunged into liquid nitrogen. All embryos were subsequently thawed and rehydrated either directly into holding media or into holding media containing 0.2 m sucrose and were incubated for 5 or 10 min before being transferred to holding media before transfer to recipients. Although the survival rate of the embryos immediately after thawing was high (OPS 20%/40% ethanediol resulted in 97% and 100% survival for Day 6 and Day 7 embryos, respectively; OPS 10%/20% ethanediol resulted in 90% and 70% survival for Day 6 and Day 7 embryos, respectively), after 2 h in culture, survival rates had decreased to 46% and 53% for Day 6 and Day 7 embryos, respectively, using OPS 10%/20% and 53% and 63% for Day 6 and Day 7 embryos, respectively, using OPS 20%/40%; however, none of the embryos transferred resulted in a viable fetus. A further 63 embryos (Day 6: n = 31; Day 7: n = 16; Day 8: n = 16) were subsequently exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 m sucrose + 0.375 m glucose + 3% polyethylene glycol) in three steps and after loading into 0.25 mL straws were plunged into liquid nitrogen. However, a much greater percentage of the Day 7 and Day 8 embryos (43.8% and 81.2% respectively) were fractured or torn after warming and none of the 12 intact embryos transferred resulted in a pregnancy. Better survival rates immediately after thawing and rehydration were obtained with the smaller Day 6 embryos (94%), which resulted in a total of eight fetuses from the 21 embryos transferred.
Age-related changes in the oestrous cycle and reproductive hormones in senescence-accelerated mouse
Authors: Yuan Ma , Wen-Xia Zhou , Jun-Ping Cheng , Yong-Xiang Zhang
Published: May 2005

To investigate age-related changes in the oestrous cycle and reproductive hormone levels in senescence-accelerated mouse (SAM), we examined these parameters in 3-, 5-, 7-, 9- and 11-month-old female SAM-prone/8 (SAMP8) and SAM-resistant/1 (SAMR1) strains. Levels of β-endorphin (β-EP) and substance P (SP) in the hypothalamus were also measured. The oestrous cycle and dioestrus of 9-month-old SAMP8 mice were significantly prolonged compared with age-matched SAMR1 mice. Furthermore, the concentration of serum oestradiol was lower and the level of pituitary luteinising hormone was higher in SAMP8 mice compared with SAMR1 mice. This characterises the hypothalamus–pituitary–ovary (HPO) axis of the SAMP8 strain as hypergonadotropic–hypogonad. The levels of β-EP and SP in the SAMP8 hypothalamus were lower than in the SAMR1 hypothalamus. These results indicate that the function of the HPO axis in SAMP8 mice declines early and this may be attributed, in part, to the decline in β-EP and SP concentrations in the hypothalamus.
Abundance of ADAM-8, -9, -10, -12, -15 and -17 and ADAMTS-1 in mouse uterus during the oestrous cycle
Authors: Kim Jiyoung , Kim Haekwon , Lee Joon Yeong , Choi Young Min , Lee Su-Jae , Lee Seung-Jae
Published: May 2005

The aim of the present study was to determine whether a disintegrin and metalloproteinase (ADAM)-8, -9, -10, -12, -15 and -17 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 are involved in the remodelling process of the mouse uterus during the oestrous cycle. The mRNA expression of ADAM was observed in all uterine tissues throughout the entire cycle. The levels of ADAM-8 mRNA were maximal at pro-oestrus, whereas the expression of ADAM-9 and ADAMTS-1 mRNA was maximal at oestrus. The minimum mRNA level of all ADAM genes always occurred at dioestrus. The mRNA levels of ADAM-10, -12, -15 and -17 did not vary significantly, regardless of the stage of the oestrous cycle. Immunoblot analyses demonstrated the presence of all ADAM proteins throughout the cycle. In terms of protein intensities, ADAM-8, -12 and -17 were maximal at pro-oestrus, whereas ADAM-10 and ADAMTS-1 were maximal at metoestrus and ADAM-9 was maximal at oestrus. Regardless of the ADAM species, minimal protein expression always occurred at dioestrus. Immunohistochemical studies showed ADAM protein expression in luminal and glandular epithelial layers, but not in the stromal layer. Moreover, ADAM proteins were found to be heterogeneously localised and their individual localisations depended on the stage of the oestrous cycle. From these observations, we suggest that the ADAM genes play an important role in mouse uterine tissue remodelling during the oestrous cycle.
Comparison of two approaches to nuclear transfer in the bovine: hand-made cloning with modifications and the conventional nuclear transfer technique
Authors: Tecirlioglu R. Tayfur , Cooney Melissa A. , Lewis Ian M. , Korfiatis Natasha A. , Hodgson Renee , Ruddock Nancy T. , Vajta Gábor , Downie Shara , Trounson Alan O. , Holland Michael K. , French Andrew J.
Published: May 2005

The aim of the present study was to compare the in vitro and in vivo developmental competence of hand-made cloning (HMC) embryos with the conventional nuclear transfer (NT) method using five somatic cell lines and in vitro-fertilised (IVF; control) embryos. Modifications to the HMC procedure included fusion efficiency optimisation, effect of cytoplasmic volume and cloned embryo aggregation. The developmental competence of blastocysts from each of the treatment groups and cell lines used was assessed following transfer to 345 recipients. Vitrification was also used to enable management of recipient resources and to assess the susceptibility of membranes to cryopreservation following zona removal. Increasing cytoplasmic volume to 150% or aggregating two embryos improved the blastocyst development rate and increased the total cell number. Although HMC embryo transfers established a significantly higher pregnancy rate on Day 30 than fresh IVF or NT embryo transfers, the overall outcome in terms of cloned live births derived from either fresh or vitrified/thawed HMC or NT embryo transfers across the five cell lines did not differ. The birth and continued survival of clones produced with HMC technology with equivalent efficiency to NT shows that it can be used as an alternative method for the generation of cloned offspring in the bovine.
Comparison of gene expression in individual preimplantation bovine embryos produced by in vitro fertilisation or somatic cell nuclear transfer
Authors: Camargo Luiz Sergio de A. , Powell Anne M. , Filho Vicente R. do Vale , Wall Robert J.
Published: May 2005

In vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) have been implicated in a variety of developmental abnormalities. Aberrant gene expression is likely to account for much of the diminished viability and developmental abnormalities observed. In the present study, the expression of multiple genes in IVF and SCNT bovine blastocyst-stage embryos were evaluated and compared with in vivo-produced embryos. Eleven genes expressed at and following maternal–zygotic transcription transition were evaluated in individual blastocysts by real-time polymerase chain reaction following RNA amplification. A subset of those genes was also evaluated in individual IVF and SCNT eight-cell embryos. A fibroblast-specific gene, expressed by nuclear donor cells, was also evaluated in IVF and SCNT embryos. The observed gene expression pattern at the eight-cell stage was not different between IVF and SCNT embryos (P > 0.05). In vitro fertilisation and SCNT blastocyst expression was lower (P < 0.01) for all genes compared with their in vivo-produced counterparts, except for lactate dehydrogenase isoenzyme A (P < 0.001). The patterns of gene expression of the IVF and SCNT blastocysts were indistinguishable. Neither SCNT eight-cell nor blastocyst-stage embryos expressed the gene used as a fibroblast marker (collagen VIα1). For the genes evaluated, the level of expression was influenced more by the environment than by the method used to produce the embryos. These results support the notion that if developmental differences observed in IVF- and SCNT-produced fetuses and neonates are the result of aberrant gene expression during the preimplantation stage, those differences in expression are subtle.
Experimentally induced subnormal or normal luteal phases in sheep: reproductive hormone profiles and uterine sex steroid receptor expression
Authors: Tasende C. , Forsberg M. , Rodríguez-Piñón M. , Acuña S. , Garófalo E. G.
Published: May 2005

This study investigated if ewes expected to have subnormal luteal phases (SNLP) present a different pattern of uterine oestrogen receptor (ER) and progesterone receptor (PR) expression at the expected time of premature luteolysis. The concentrations of uterine ER, PR and ERα mRNA, and the steroid ovarian hormone were determined in anoestrous ewes treated with either gonadotrophin-releasing hormone (GnRH) to develop a SNLP (n = 16), or progesterone + GnRH to develop a normal luteal phase (NLP; n = 16). The ER, PR and ERα mRNA concentrations were measured using binding and solution hybridisation assays, while the hormone level concentration was measured by radioimmunoassay. In all ewes, a luteinising hormone- and follicle-stimulating hormone-synchronised surge was found. The SNLP group had lower preovulatory oestradiol levels than the NLP group. On Day 5, the SNLP group had lower progesterone levels, and higher uterine ER, PR and ERα mRNA concentrations than the NLP group. While in the SNLP group the receptor expression increased from Days 1 to 5, in the NLP group the receptor expression decreased. The results suggest that the induction of steroid receptor expression in the uterus and the hormonal environment found in the experimental SNLP group at the expected time of premature luteolysis may be involved in the mechanisms causing SNLP.
Effects of 2,2',5,5'-tetrachlorobiphenyl (PCB52) on migration of chicken primordial germ cells
Authors: Zhang Yixiang , Jin Xiumei , Han Haitang , Li Zandong
Published: May 2005

Polychlorinated biphenyls cause developmental and physiological anomalies in the reproductive system. This study investigated the effects of 2,2′,5,5′-tetrachlorobiphenyl (PCB52), which can produce oestrogenic effects on the homeostasis of chicken primordial germ cells from the initial stage until completion of their settlement in the gonadal primordium. The blastoderm of chicken embryos was injected with 1 μL PCB52 (10 µmol/L) and oestradiol (100 µmol/L) before incubation, and the number of primordial germ cells was determined during their migration and development. The number of primordial germ cells in germinal crescents in PCB52-treated groups was slightly decreased (P = 0.068), but it was reduced significantly at stages 13–15 and 28–30 (P < 0.01, respectively) compared with controls. No obvious effects on primordial germ cell migration were observed with oestradiol treatments. The present results suggest that the influence of PCB52 on chicken primordial germ cell migration and proliferation may be via its toxic effect, not its oestrogen-mimicking effect, and provide information on the sensitivity of primordial germ cells to the direct action of PCB52.
A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection
Authors: Morris Emma C. , Tsallios Aristotle , Bendle Gavin M. , Xue Shao-an , Stauss Hans J.
Published: May 2005

Adoptive transfer of antigen-specific CD4+and CD8+T cells is one of the most efficient forms of cancer immunotherapy. However, the isolation of antigen-specific CD4+T cells is limited because only few tumor-associated helper epitopes are identified. Here, we used T cell antigen receptor gene transfer to target CD4+T cells against an MHC class I-presented epitope of a model tumor antigen. IFN-γ-producing CD4+T cells were unable to expandin vivoand to provide help for tumor rejection. In contrast, CD4+T cells producing high levels of IL-2 expandedin vivo, provided help for cytotoxic T lymphocyte-mediated tumor rejection, and developed T cell memory. The data demonstratein vivosynergy between T cell antigen receptor-transduced CD4+and CD8+T cells specific for the same epitope resulting in long-term tumor protection.
Reassessing Coxcatlan Cave and the early history of domesticated plants in Mesoamerica
Authors: Smith Bruce D.
Published: May 2005

Reanalysis and direct accelerator mass spectrometry radiocarbon dating of the cucurbit assemblage from Coxcatlan Cave provide information on the timing and sequence of the initial appearance of three domesticated plants in the Tehuacán Valley (Puebla, Mexico) and allow reassessment of the overall temporal context of plant domestication in Mexico.
Cucurbita pepo
is the earliest documented domesticate in the cave, dating to 7,920 calibrated calendrical (cal) years B.P. The bottle gourd (
Lagenaria siceraria
) is dated at 7,200 cal years B.P.
Cucurbita argyrosperma
does not appear until 2,065 cal years B.P. The earlier identification of
Cucurbita moschata
specimens is not confirmed. Seventy-one radiocarbon dates, including 23 accelerator mass spectrometry dates on cucurbits, provide ample evidence of postdepositional vertical displacement of organic materials in the western half of Coxcatlan Cave, but they also indicate that the eastern half of the cave was largely undisturbed.
Processing of VEGF-A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors
Authors: Lee Sunyoung , Jilani Shahla M. , Nikolova Ganka V. , Carpizo Darren , Iruela-Arispe M. Luisa
Published: May 2005

Vascular endothelial growth factor (VEGF) is a critical mediator of blood vessel formation during development and in pathological conditions. In this study, we demonstrate that VEGF bioavailability is regulated extracellularly by matrix metalloproteinases (MMPs) through intramolecular processing. Specifically, we show that a subset of MMPs can cleave matrix-bound isoforms of VEGF, releasing soluble fragments. We have mapped the region of MMP processing, have generated recombinant forms that mimic MMP-cleaved and MMP-resistant VEGF, and have explored their biological impact in tumors. Although all forms induced similar VEGF receptor 2 phosphorylation levels, the angiogenic outcomes were distinct. MMP-cleaved VEGF promoted the capillary dilation of existent vessels but mediated a marginal neovascular response within the tumor. In contrast, MMP-resistant VEGF supported extensive growth of thin vessels with multiple and frequent branch points. Our findings support the view that matrix-bound VEGF and nontethered VEGF provide different signaling outcomes. These findings reveal a novel aspect in the regulation of extracellular VEGF that holds significance for vascular patterning.
Junctional membrane inositol 1,4,5-trisphosphate receptor complex coordinates sensitization of the silent EGF-induced Ca2+ signaling
Authors: Hur Eun-Mi , Park Yong-Soo , Huh Yang Hoon , Yoo Seung Hyun , Woo Kyung-Chul , Choi Bo-Hwa , Kim Kyong-Tai
Published: May 2005

Ca2+ is a highly versatile intracellular signal that regulates many different cellular processes, and cells have developed mechanisms to have exquisite control over Ca2+ signaling. Epidermal growth factor (EGF), which fails to mobilize intracellular Ca2+ when administrated alone, becomes capable of evoking [Ca2+]i increase and exocytosis after bradykinin (BK) stimulation in chromaffin cells. Here, we provide evidence that this sensitization process is coordinated by a macromolecular signaling complex comprised of inositol 1,4,5-trisphosphate receptor type I (IP3R1), cAMP-dependent protein kinase (PKA), EGF receptor (EGFR), and an A-kinase anchoring protein, yotiao. The IP3R complex functions as a focal point to promote Ca2+ release in two ways: (1) it facilitates PKA-dependent phosphorylation of IP3R1 in response to BK-induced elevation of cAMP, and (2) it couples the plasmalemmal EGFR with IP3R1 at the Ca2+ store located juxtaposed to the plasma membrane. Our study illustrates how the junctional membrane IP3R complex connects different signaling pathways to define the fidelity and specificity of Ca2+ signaling.
Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases
Authors: Luo Shouqing , Vacher Coralie , Davies Janet E. , Rubinsztein David C.
Published: May 2005

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. Mutant htt toxicity is exposed after htt cleavage by caspases and other proteases release NH2-terminal fragments containing the polyQ expansion. Here, we show htt interacts and colocalizes with cdk5 in cellular membrane fractions. Cdk5 phosphorylates htt at Ser434, and this phosphorylation reduces caspase-mediated htt cleavage at residue 513. Reduced mutant htt cleavage resulting from cdk5 phosphorylation attenuated aggregate formation and toxicity in cells expressing the NH2-terminal 588 amino acids (htt588) of mutant htt. Cdk5 activity is reduced in the brains of HD transgenic mice compared with controls. This result can be accounted for by the polyQ-expanded htt fragments reducing the interaction between cdk5 and its activator p35. These data predict that the ability of cdk5 phosphorylation to protect against htt cleavage, aggregation, and toxicity is compromised in cells expressing toxic fragments of htt.
FIAT represses ATF4-mediated transcription to regulate bone mass in transgenic mice
Authors: Yu Vionnie W.C. , Ambartsoumian Gourgen , Verlinden Lieve , Moir Janet M. , Prud'homme Josée , Gauthier Claude , Roughley Peter J. , St-Arnaud René
Published: May 2005

We report the characterization of factor inhibiting activating transcription factor 4 (ATF4)–mediated transcription (FIAT), a leucine zipper nuclear protein. FIAT interacted with ATF4 to inhibit binding of ATF4 to DNA and block ATF4-mediated transcription of the osteocalcin gene in vitro. Transgenic mice overexpressing FIAT in osteoblasts also had reduced osteocalcin gene expression and decreased bone mineral density, bone volume, mineralized volume, trabecular thickness, trabecular number, and decreased rigidity of long bones. Mineral homeostasis, osteoclast number and activity, and osteoblast proliferation and apoptosis were unchanged in transgenics. Expression of osteoblastic differentiation markers was largely unaffected and type I collagen synthesis was unchanged. Mineral apposition rate was reduced in transgenic mice, suggesting that the lowered bone mass was due to a decline in osteoblast activity. This cell-autonomous decrease in osteoblast activity was confirmed by measuring reduced alkaline phosphatase activity and mineralization in primary osteoblast cultures. These results show that FIAT regulates bone mass accrual and establish FIAT as a novel transcriptional regulator of osteoblastic function.
A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice
Authors: Kieran Dairin , Hafezparast Majid , Bohnert Stephanie , Dick James R.T. , Martin Joanne , Schiavo Giampietro , Fisher Elizabeth M.C. , Greensmith Linda
Published: May 2005

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by motoneuron degeneration and muscle paralysis. Although the precise pathogenesis of ALS remains unclear, mutations in Cu/Zn superoxide dismutase (SOD1) account for ∼20–25% of familial ALS cases, and transgenic mice overexpressing human mutant SOD1 develop an ALS-like phenotype. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. In Legs at odd angles (Loa) mice, mutations in the motor protein dynein are associated with axonal transport defects and motoneuron degeneration. Here, we show that retrograde axonal transport defects are already present in motoneurons of SOD1G93A mice during embryonic development. Surprisingly, crossing SOD1G93A mice with Loa/+ mice delays disease progression and significantly increases life span in Loa/SOD1G93A mice. Moreover, there is a complete recovery in axonal transport deficits in motoneurons of these mice, which may be responsible for the amelioration of disease. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS.
Endoplasmic reticulum stress modulates the response of myelinating oligodendrocytes to the immune cytokine interferon-γ
Authors: Lin Wensheng , Harding Heather P. , Ron David , Popko Brian
Published: May 2005

I*nterferon-γ (IFN-γ) is believed to contribute to immune-mediated demyelinating disorders by targeting the myelin-producing oligodendrocyte, a cell known to be highly sensitive to the disruption of protein synthesis and to the perturbation of the secretory pathway. We found that apoptosis induced by IFN-γ in cultured rat oligodendrocytes was associated with endoplasmic reticulum (ER) stress. ER stress also accompanied oligodendrocyte apoptosis and hypomyelination in transgenic mice that inappropriately expressed IFN-γ in the central nervous system (CNS). Compared with a wild-type genetic background, the enforced expression of IFN-γ in mice that were heterozygous for a loss of function mutation in pancreatic ER kinase (PERK) dramatically reduced animal survival, promoted CNS hypomyelination, and enhanced oligodendrocyte loss. PERK encodes an ER stress–inducible kinase that phosphorylates eukaryotic translation initiation factor 2α and specifically maintains client protein homeostasis in the stressed ER. Therefore, the hypersensitivity of PERK+/− mice to IFN-γ implicates ER stress in demyelinating disorders that are induced by CNS inflammation.
Structurally and functionally unique complexins at retinal ribbon synapses
Authors: Reim Kerstin , Wegmeyer Heike , Brandstätter Johann Helmut , Xue Mingshan , Rosenmund Christian , Dresbach Thomas , Hofmann Kay , Brose Nils
Published: May 2005

Ribbon synapses in retinal sensory neurons maintain large pools of readily releasable synaptic vesicles. This allows them to release several hundreds of vesicles per second at every presynaptic release site. The molecular components that cause this high transmitter release efficiency of ribbon synapses are unknown. In the present study, we identified and characterized two novel vertebrate complexins (CPXs), CPXs III and IV, that are the only CPX isoforms present in retinal ribbon synapses. CPXs III and IV are COOH-terminally farnesylated, and, like CPXs I and II, bind to SNAP receptor complexes. CPXs III and IV can functionally replace CPXs I and II, and their COOH-terminal farnesylation regulates their synaptic targeting and modulatory function in transmitter release. The novel CPXs III and IV may contribute to the unique release efficacy of retinal sensory neurons.
SEC18/NSF-independent, protein-sorting pathway from the yeast cortical ER to the plasma membrane
Authors: Jüschke Christoph , Wächter Andrea , Schwappach Blanche , Seedorf Matthias
Published: May 2005

Classic studies of temperature-sensitive secretory (sec) mutants have demonstrated that secreted and plasma membrane proteins follow a common SEC pathway via the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles to the cell periphery. The yeast protein Ist2p, which is synthesized from a localized mRNA, travels from the ER to the plasma membrane via a novel route that operates independently of the formation of coat protein complex II–coated vesicles. In this study, we show that the COOH-terminal domain of Ist2p is necessary and sufficient to mediate SEC18-independent sorting when it is positioned at the COOH terminus of different integral membrane proteins and exposed to the cytoplasm. This domain functions as a dominant plasma membrane localization determinant that overrides other protein sorting signals. Based on these observations, we suggest a local synthesis of Ist2p at cortical ER sites, from where the protein is sorted by a novel mechanism to the plasma membrane.
Electrophysiological Characterization of the Arabidopsis avrRpt2-Specific Hypersensitive Response in the Absence of Other Bacterial Signals
Authors: Pike Sharon M. , Zhang Xue-Cheng , Gassmann Walter
Published: May 2005

The hypersensitive response (HR) is defined as rapid cell collapse at the infection site and often accompanies plant resistance. The physiological processes leading to HR are not well understood. Here, we report an electrophysiological characterization of bacterial HR caused by a single avirulence gene in the absence of other bacterial signals. We used dexamethasone (dex)-inducible transgenic Arabidopsis (Arabidopsis thaliana) plants containing the avrRpt2 gene from Pseudomonas syringae pv tomato. Membrane depolarization in these plants began 1 to 1.5 h after dex application, hours before electrolyte leakage. Progressive depolarization was a sensitive early indicator of HR that occurred only in Arabidopsis leaf cells expressing both avrRpt2 and a functional RPS2 gene. Hyperpolarization of fully depolarized membranes by fusicoccin, a fungal toxin that activates the H+-ATPase, indicates that depolarization did not result from a nonfunctional pump or leaky membranes. Depolarization and electrolyte leakage were inhibited in RPS2 plants by the calcium channel blocker LaCl3, highly correlating these events and suggesting that Ca2+ entry into cells is required for both. Also correlated were inhibition of depolarization, electrolyte leakage, and HR following salicylic acid pretreatment. In salicylic acid-pretreated RPS2 seedlings, avrRpt2 transcript was produced after dex treatment. However, AvrRpt2 protein accumulation was greatly reduced, suggesting a possible mechanism for inhibition of HR in plants with induced resistance. This experimental system is a very sensitive assay that lends itself to the dissection of physiological processes leading to HR in plants, and provides a baseline for future research within a genetic framework.
Root Border-Like Cells of Arabidopsis. Microscopical Characterization and Role in the Interaction with Rhizobacteria
Authors: Vicré Maïté , Santaella Catherine , Blanchet Sandrine , Gateau Aurélien , Driouich Azeddine
Published: May 2005

Plant roots of many species produce thousands of cells that are released daily into the rhizosphere. These cells are commonly termed border cells because of their major role in constituting a biotic boundary layer between the root surface and the soil. In this study, we investigated the occurrence and ultrastructure of such cells in Arabidopsis (Arabidopsis thaliana) using light and electron microscopy coupled to high-pressure freezing. The secretion of cell wall molecules including pectic polysaccharides and arabinogalactan-proteins (AGPs) was examined also using immunofluorescence microscopy and a set of anticarbohydrate antibodies. We show that root tips of Arabidopsis seedlings released cell layers in an organized pattern that differs from the rather randomly dispersed release observed in other plant species studied to date. Therefore, we termed such cells border-like cells (BLC). Electron microscopical results revealed that BLC are rich in mitochondria, Golgi stacks, and Golgi-derived vesicles, suggesting that these cells are actively engaged in secretion of materials to their cell walls. Immunocytochemical data demonstrated that pectins as well as AGPs are among secreted material as revealed by the high level of expression of AGP-epitopes. In particular, the JIM13-AGP epitope was found exclusively associated with BLC and peripheral cells in the root cap region. In addition, we investigated the function of BLC and root cap cell AGPs in the interaction with rhizobacteria using AGP-disrupting agents and a strain of Rhizobium sp. expressing a green fluorescent protein. Our findings demonstrate that alteration of AGPs significantly inhibits the attachment of the bacteria to the surface of BLC and root tip.
Loss-of-Function Mutations of ROOT HAIR DEFECTIVE3 Suppress Root Waving, Skewing, and Epidermal Cell File Rotation in Arabidopsis
Authors: Yuen Christen Y.L. , Sedbrook John C. , Perrin Robyn M. , Carroll Kathleen L. , Masson Patrick H.
Published: May 2005

Wild-type Arabidopsis (Arabidopsis thaliana L. Heynh.) roots growing on a tilted surface of impenetrable hard-agar media adopt a wave-like pattern and tend to skew to the right of the gravity vector (when viewed from the back of the plate through the medium). Reversible root-tip rotation often accompanies the clockwise and counterclockwise curves that form each wave. These rotations are manifested by epidermal cell file rotation (CFR) along the root. Loss-of-function alleles of ROOT HAIR DEFECTIVE3 (RHD3), a gene previously implicated in the control of vesicle trafficking between the endoplasmic reticulum and the Golgi compartments, resulted in an almost complete suppression of epidermal CFR, root skewing, and waving on hard-agar surfaces. Several other root hair defective mutants (rhd2-1, rhd4-1, and rhd6-1) did not exhibit dramatic alterations in these root growth behaviors, suggesting that a generalized defect in root hair formation is not responsible for the surface-dependent phenotypes of rhd3. However, similar alterations in root growth behavior were observed in a variety of mutants characterized by defects in cell expansion (cob-1, cob-2, eto1-1, eto2-1, erh2-1, and erh3-1). The erh2-1 and rhd3-1 mutants differed from other anisotropic cell expansion mutants, though, by an inability to respond to low doses of the microtubule-binding drug propyzamide, which normally causes enhanced left-handed CFR and right skewing. We hypothesize that RHD3 may control epidermal CFR, root skewing, and waving on hard-agar surfaces by regulating the traffic of wall- or plasma membrane-associated determinants of anisotropic cell expansion.
Mutations Affecting Starch Synthase III in Arabidopsis Alter Leaf Starch Structure and Increase the Rate of Starch Synthesis
Authors: Zhang Xiaoli , Myers Alan M. , James Martha G.
Published: May 2005

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.
Involvement of Phycobilisome Diffusion in Energy Quenching in Cyanobacteria
Authors: Joshua Sarah , Bailey Shaun , Mann Nicholas H. , Mullineaux Conrad W.
Published: May 2005

Nonphotochemical quenching (NPQ) of excitation energy is a well-established phenomenon in green plants, where it serves to protect the photosynthetic apparatus from photodamage under excess illumination. The induction of NPQ involves a change in the function of the light-harvesting apparatus, with the formation of quenching centers that convert excitation energy into heat. Recently, a comparable phenomenon was demonstrated in cyanobacteria grown under iron-starvation. Under these conditions, an additional integral membrane chlorophyll-protein, IsiA, is synthesized, and it is therefore likely that IsiA is required for NPQ in cyanobacteria. We have previously used fluorescence recovery after photobleaching to show that phycobilisomes diffuse rapidly on the membrane surface, but are immobilized when cells are immersed in high-osmotic strength buffers, apparently because the interaction between phycobilisomes and reaction centers is stabilized. Here, we show that when cells of the cyanobacterium Synechocystis sp. PCC 6803 subjected to prolonged iron-deprivation are immersed in 1 m phosphate buffer, NPQ can still be induced as normal by high light. However, the formation of the quenched state is irreversible under these conditions, suggesting that it involves the coupling of free phycobilisomes to an integral-membrane complex, an interaction that is stabilized by 1 m phosphate. Fluorescence spectra are consistent with this idea. Fluorescence recovery after photobleaching measurements confirm that the induction of NPQ in the presence of 1 m phosphate is accompanied by immobilization of the phycobilisomes. We propose as a working hypothesis that a major component of the fluorescence quenching observed in iron-starved cyanobacteria arises from the coupling of free phycobilisomes to IsiA.
Rapid Array Mapping of Circadian Clock and Developmental Mutations in Arabidopsis
Authors: Hazen Samuel P. , Borevitz Justin O. , Harmon Frank G. , Pruneda-Paz Jose L. , Schultz Thomas F. , Yanovsky Marcelo J. , Liljegren Sarah J. , Ecker Joseph R. , Kay Steve A.
Published: May 2005

Classical forward genetics, the identification of genes responsible for mutant phenotypes, remains an important part of functional characterization of the genome. With the advent of extensive genome sequence, phenotyping and genotyping remain the critical limiting variables in the process of map-based cloning. Here, we reduce the genotyping problem by hybridizing labeled genomic DNA to the Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 GeneChip. Genotyping was carried out on the scale of detecting greater than 8,000 single feature polymorphisms from over 200,000 loci in a single assay. By combining this technique with bulk segregant analysis, several high heritability development and circadian clock traits were mapped. The mapping accuracy using bulk pools of 26 to 100 F2 individuals ranged from 0.22 to 1.96 Mb of the mutations revealing mutant alleles of EARLY FLOWERING 3, EARLY FLOWERING 4, TIMING OF CAB EXPRESSION 1, and ASYMMETRIC LEAVES 1. While direct detection of small mutations, such as an ethyl-methane sulfonate derived single base substitutions, is limited by array coverage and sensitivity, large deletions such as those that can be caused by fast neutrons are easily detected. We demonstrate this by resolving two deletions, the 77-kb flavin-binding, kelch repeat, f-box 1 and the 7-kb cryptochrome2-1 deletions, via direct hybridization of mutant DNA to ATH1 expression arrays.
A Molecular-Genetic Study of the Arabidopsis Toc75 Gene Family
Authors: Baldwin Amy , Wardle Anthony , Patel Ramesh , Dudley Penny , Park Soon Ki , Twell David , Inoue Kentaro , Jarvis Paul
Published: May 2005

Toc75 (translocon at the outer envelope membrane of chloroplasts, 75 kD) is the protein translocation channel at the outer envelope membrane of plastids and was first identified in pea (Pisum sativum) using biochemical approaches. The Arabidopsis (Arabidopsis thaliana) genome contains three Toc75-related sequences, termed atTOC75-I, atTOC75-III, and atTOC75-IV, which we studied using a range of molecular, genetic, and biochemical techniques. Expression of atTOC75-III is strongly regulated and at its highest level in young, rapidly expanding tissues. By contrast, atTOC75-IV is expressed uniformly throughout development and at a much lower level than atTOC75-III. The third sequence, atTOC75-I, is a pseudogene that is not expressed due to a gypsy/Ty3 transposon insertion in exon 1, and numerous nonsense, frame-shift, and splice-junction mutations. The expressed genes, atTOC75-III and atTOC75-IV, both encode integral envelope membrane proteins. Unlike atToc75-III, the smaller atToc75-IV protein is not processed upon targeting to the envelope, and its insertion does not require ATP at high concentrations. The atTOC75-III gene is essential for viability, since homozygous atToc75-III knockout mutants (termed toc75-III) could not be identified, and aborted seeds were observed at a frequency of approximately 25% in the siliques of self-pollinated toc75-III heterozygotes. Homozygous toc75-III embryos were found to abort at the two-cell stage. Homozygous atToc75-IV knockout plants (termed toc75-IV) displayed no obvious visible phenotypes. However, structural abnormalities were observed in the etioplasts of toc75-IV seedlings and atTOC75-IV overexpressing lines, and toc75-IV plants were less efficient at deetiolation than wild type. These results suggest some role for atToc75-IV during growth in the dark.
The measurement of racial discrimination: the policy use of statistics
Authors: Simon Patrick
Published: May 2005

Most ‘multicultural societies’ in the world attempt to act against racial discrimination and some, admittedly less numerous, have adopted pro‐active equality policies based on statistical monitoring. Intensive use of statistics is a requirement for action against indirect discrimination, a legal concept imported into European Union countries since 2000. However, design and collection of statistical data providing information on racial discrimination calls for the establishment of a technical apparatus that raises issues of political strategy and action methodology. Using the results of a comparative study of the statistics used in anti‐discrimination policy in the USA, the UK, Canada, and Australia, this analysis compares the various categories and modes of collection by setting them in context in order to reveal the compromises made between the legal and political imperatives of the struggle against discrimination and the aim of identity recognition within the multicultural project.
The role of statistics on ethnic origin and ‘race’ in Canadian anti‐discrimination policy
Authors: Potvin Maryse
Published: May 2005

This article sets the history of the development of Canadian anti‐discrimination policy and of legal and practical definition of statistical categories relating to ‘race’ and ethnic origin within a strategy of substantive equality and action against discrimination. It is shown that the elaboration of such categories, their development, changing nomenclatures and the debates to which they have given rise are set within the history of power relations between the various majority and minority groups in Canada. The article also stresses that use of social statistics, which is now recognised to be basic and even essential in courts of law as well as in the development of legislation and policy, bears witness to growing articulation in Canada, over the last thirty or so years, between research, the law, and anti‐discrimination policies.
Mobilising statistical powers for action against discrimination: the case of the United Kingdom*
Authors: Stavo‐Debauge Joan
Published: May 2005

The article proposes to shed light on the links between anti‐discrimination law, regarded in terms of its implementation and its policy development, and statistics by showing how initial distrust, among researchers as well as among activists and policy makers, gives way to confidence in the apparatus of ethnic and/or racial categories. The article first considers legal provisions and the constraints entailed by their operationalisation, with particular reference to the central character of ethnic monitoring in British anti‐discrimination policy and to the latter's multicultural turn. The author then addresses the justifications and criticisms that accompanied the development of the ethnic categories included in the 1991 and 2001 censuses and contributed to their renewal.
From sword to plowshare: using race for discrimination and antidiscrimination in the United States
Authors: Morning Ann , Sabbagh Daniel
Published: May 2005

This article addresses the question of how the United States' policies of antidiscrimination drew on official racial categories that were traditionally used explicitly for discriminatory purposes. After briefly recounting the history of official racial classification practices in the United States and their relation to racist laws and practices, we describe the development of legal prohibitions on racial discrimination, culminating in the civil rights movement of the 1950s, 60s, and 70s. This leads to an examination of how statistical data on race are used to implement civil rights law, before outlining contemporary official racial categories. Finally, we assess the debates that have arisen concerning the collection of racial statistics for the purposes of antidiscrimination enforcement. In conclusion, we argue that, more than any autonomous political challenge to the principle of government race classification, what has emerged powerfully in recent years is an awareness of tensions between the needs of the civil rights enforcement machinery and the emerging claim of Americans' “right” to self‐define racially as they see fit – in short, between the politics of distribution and the politics of recognition.
Monitoring ethnic minorities in the Netherlands
Authors: Guiraudon Virginie , Phalet Karen , Ter Wal Jessika
Published: May 2005

The article first summarises the history of ethnic minority policy in the Netherlands and the development of the ‘ethnic minority’ and ‘allochthonous’ categories, which are peculiar in comparative perspective in emphasising socio‐economic disadvantage as a constitutive dimension of minority status and in setting the minority question within the broader Dutch political principle of ‘pillarisation’. The article then examines the use of statistics in public policy, in a context where the national census has been discontinued since 1971, focusing more specifically on the case of education, where major statistical efforts have been devoted to identifying patterns of disadvantage and integration. Finally, the article briefly examines current debates on the situation of ethnic minorities in the Netherlands in the context of growing questioning of established Dutch models of minority policy.
Building American citizenship: a matter of rights or of races?
Authors: Bessone Magali
Published: May 2005

As proclaimed in the Declaration of Independence, US citizenship is based on natural law. However, the very language of the Declaration also points to the presence of another ideological dimension, alongside natural law and the social contract. The Declaration gives its allegiance to “nature” and to “nature's God”, which are not simply the rational basis of moral laws to be enshrined in positive law, but also express anorderreflective of divine will, which establishes distinctions and hierarchies even within such “nature”, including human nature. The alliance, or even the fusion, of the two inherent dimensions of the idea of nature – the ultimate reference underwriting the validity of certain protective principles, as well as the legitimising basis of visible inequalities – is undoubtedly the main distinctive feature of American political philosophy. This article proposes to analyse the intertwining of the two apparently contradictory principles of universalism and “racialism” and the role of this intricate relationship in the birth of the American nation by following their trace from the foundational language of Thomas Jefferson to the subversive critique of W.E.B. Du Bois.