Granular aluminium nanojunction fluxonium qubit.

Mesoscopic Josephson junctions, consisting of overlapping superconducting electrodes separated by a nanometre-thin oxide layer, provide a precious source of nonlinearity for superconducting quantum circuits. Here we show that in a fluxonium qubit, the role of the Josephson junction can also be played by a lithographically defined, self-structured granular aluminium nanojunction: a superconductor-insulator-superconductor Josephson junction obtained in a single-layer, zero-angle evaporation. The measured spectrum of the resulting qubit, which we nickname gralmonium, is indistinguishable from that of a standard fluxonium. Remarkably, the lack of a mesoscopic parallel plate capacitor gives rise to an intrinsically large granular aluminium nanojunction charging energy in the range of tens of gigahertz, comparable to its Josephson energy. We measure coherence times in the microsecond range and we observe spontaneous jumps of the value of the Josephson energy on timescales from milliseconds to days, which offers a powerful diagnostics tool for microscopic defects in superconducting materials.

Involvement of superoxide in malaoxon-induced toxicity in primary cultures of cortical neurons.

Organophosphorus compounds (OP) represent a class of insecticides that are used most globally. The neurotoxic effects are attributed mainly to acetylcholinesterase (AChE) enzyme inhibition, which is responsible for cholinergic manifestations in individuals acutely exposed to OP. However, AChE inhibition alone cannot account for the wide range of symptoms that were reported following OP exposures. In agreement with this, evidence shows that non-cholinergic events may be mechanistically linked to OP-induced neurotoxicity. The aim of this study was to investigate the potential occurrence of oxidative stress as a critical step in the toxicity induced by the OP malaoxon(MAL) using primary cultures of mouse cortical neurons, as well as to distinguish MAL-induced oxidative stress and cell toxicity from an action on AChE blockade. Primary cultures of mouse cortical neurons were treated with MAL (0.01; 0.1; 1; 10; or 100 µM) at varying time points (1, 3, 6, 24, 48, or 144 hr) and the following biochemical parameters determined including cell viability, AChE activity, and superoxide production. MAL significantly reduced cell viability in a concentration- and time-dependent manner. Of note, 1 µM MAL significantly inhibited (approximately 75%) AChE activity after 48 hr incubation. Pralidoxime (PRAL) (600 µM), a classical AChE reactivator, significantly protected against MAL-induced AChE blockade; however, PRAL did not affect MAL-mediated fall in cellular viability, indicating that AChE inhibition is not necessarily correlated with insecticide-induced decrease in cell survival. MAL-induced diminished cell viability was preceded by a significant increase in superoxide anion production. The antioxidant agent ascorbic acid (AA) (200 µM), which significantly protected against MAL-induced superoxide anion production, did not alter MAL-induced AChE inhibition and significantly prevented insecticide-mediated fall in cell survival. Data show that increased superoxide anion production is an event that precedes MAL-induced cell toxicity in primary cultures of mouse cortical neurons. Based on the preventative effects of AA against MAL-mediated superoxide anion production and reduced cell viability, evidence indicates that oxidative stress represents an important step mediating MAL-induced toxicity in neurons and that AChE inhibition is not necessarily correlated with lowered cell survival noted in insecticide-exposed cells.

ConBr, A Lectin Purified from the Seeds of Canavalia brasiliensis, Protects Against Ischemia in Organotypic Culture of Rat Hippocampus: Potential Implication of Voltage-Gated Calcium Channels.

Lectins are proteins that bind cellular glycans and can modulate various neuronal functions. We have evaluated the neuroprotective effect of ConBr, a lectin purified from the seeds of Canavalia brasiliensis in a model of rat organotypic hippocampal cultures (OHCs) exposed to oxygen and glucose deprivation (OGD). OGD for 15 min followed by 24 h re-oxygenation significantly increased cell death, caused mitochondrial depolarization and increased reactive oxygen species (ROS) in CA1 region of OHCs. ConBr (0.1 µg/mL) added during the re-oxygenation period counteracted cell death, mitochondrial depolarization and overproduction of ROS induced by OGD. Moreover, ConBr restored the levels of Akt and ERK1 phosphorylation that were reduced by OGD. Modulation of intracellular Ca2+ by ConBr was evaluated in isolated hippocampal neurons loaded with the fluorescent calcium dye Fluo-4/AM. ConBr (0.1 and 1 µg/mL) reduced by 25-30 % the Ca2+ increment induced by 70 mM K+. A sub effective concentration of ConBr (0.01 µg/mL) together with a sub effective concentration of the L-type calcium channel antagonist nifedipine (0.3 µM) conferred a synergic neuroprotective effect in OHCs subjected to OGD. In conclusion, ConBr provides OHCs neuroprotection against OGD. The mechanism was not fully addressed but it may involve modulation of L-type voltage-gated Ca2+ channels by ConBr.

Atorvastatin Prevents Glutamate Uptake Reduction Induced by Quinolinic Acid Via MAPKs Signaling.

Statins have been shown to promote neuroprotection in a wide range of neurological disorders. However, the mechanisms involved in such effects of statins are not fully understood. Quinolinic acid (QA) is a neurotoxin that induces seizures when infused in vivo and promotes glutamatergic excitotoxicity in the central nervous system. The aim of this study was to evaluate the putative glutamatergic mechanisms and the intracellular signaling pathways involved in the atorvastatin neuroprotective effects against QA toxicity. Atorvastatin (10 mg/kg) treatment for 7 days prevented the QA-induced decrease in glutamate uptake, but had no effect on increased glutamate release induced by QA. Moreover, atorvastatin treatment increased the phosphorylation of ERK1 and prevented the decrease in Akt phosphorylation induced by QA. Neither atorvastatin treatment nor QA infusion altered glutamine synthetase activity or the levels of phosphorylation of p38(MAPK) or JNK1/2 during the evaluation. Inhibition of MEK/ERK signaling pathway, but not PI3K/Akt signaling, abolished the neuroprotective effect of atorvastatin against QA-induced decrease in glutamate uptake. Our data suggest that atorvastatin protective effects against QA toxicity are related to modulation of glutamate transporters via MAPK/ERK signaling pathway.

Highly palatable diet consumption increases protein oxidation in rat frontal cortex and anxiety-like behavior.

Obesity is frequently associated with consumption of high amounts of sugar and/or fat. Studies have demonstrated a high prevalence of overweight and obesity associated or not with increase rates of psychiatry disorders, in particular mood and anxiety disorders. Recent works have demonstrated an association between specific genes involved in oxidative stress metabolism and anxiety-like behavior. The aim of this study was to investigate the effect of a highly palatable diet enriched with sucrose in body fat mass composition, anxiety behavior and brain oxidative status. Twenty male Wistar rats received two different diets during four months: standard chow (SC) and highly palatable (HP). Metabolic parameters, behavioral tests and oxidative stress status were evaluated. Body fat mass, insulin sensitivity and glucose tolerance were altered in the HP group (p<0.01). The same group spends less time in light compartment and had a lower risk assessment behavior (p<0.05) but no differences were observed in the open field test habituation (p>0.05). Protein degradation, DCF and TBARS levels were not different in the hippocampus between groups; however, there were higher levels of protein degration in frontal cortex of HP groups (p<0.05), although DCF and TBARS levels don't differ from the SC group (p>0.05). In conclusion, our data suggest that the consumption of HP diet leads to an obese phenotype, increases protein oxidation in frontal cortex and appears to induce anxiety-like behavior in rats.

The lateral and dorsal neurons of Drosophila melanogaster: new insights about their morphology and function.

This chapter summarizes our present knowledge about the master clock of the fruit fly at the neuronal level. The clock is organized in distinct groups of interconnected pacemaker neurons with different functions. All of these neurons appear to communicate with one another in order to produce the species-specific activity rhythm, which is organized in morning (M) and evening (E) activity bouts. These two activity components are differentially influenced by distinct groups of pacemaker neurons reminiscent of the Pittendrigh-Daan dual oscillator model. In the original work (Grima et al. 2004; Stoleru et al. 2004), the ventrolateral (LN(v)) and dorsolateral (LN(d)) plus some dorsal groups (DN) of clock neurons have been defined as M and E cells, respectively. We further specify that the clock neurons belong to the M and E oscillators and define a more complex picture of the Drosophila brain clock.

A review of research related to blood transfusion in Canada, 2000-2002.

The former National Blood Safety Council undertook a comprehensive review of blood transfusion research in Canada for the years 2000 through 2002. Data were acquired by direct contact with agencies which support such research and by searches of the relevant websites. Total grant support increased markedly over the 3-year period, from 4.1 million dollars to 18.5 million dollars. Publicly funded granting agencies, biopharmaceutical companies, the blood services and the province of Ontario were major supporters. Much smaller amounts were granted from charitable organizations. Clinical research attracted the majority of the funding, although a larger number of projects were basic science in nature. Most research was carried out in the provinces of Ontario, Québec and British Columbia. Although we have not assessed the productivity of blood-related research, it appears that substantial amounts of funding were allocated to these projects between 2000 and 2002. These data may provide a helpful perspective to investigators in transfusion medicine elsewhere, who may also be assessing the relative priority given to this field of research in their own countries.

Effect of 2,4-dinitrophenol on the energy metabolism of cattle embryos produced by in vitro fertilization and culture.

In cattle embryos, the proportion of ATP produced by glycolysis increases following the major activation of the embryonic genome, and development to the blastocyst stage is improved in the presence of 10 microM 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, from Day 5 to Day 7 of culture. In Experiment 1 of the present study, culture of cattle embryos in the presence of 10 microM DNP from Day 5 to Day 7 stimulated development to the blastocyst stage, but had no significant effects on oxygen, pyruvate or glucose uptake, or on lactate production. In Experiment 2, culture of cattle embryos in the presence of 10 microM DNP from Day 5 to Day 7, stimulated the metabolism of [2-14C]pyruvate (a measure of Krebs cycle activity) on all of Days 5, 6 and 7, and stimulated metabolism of [5-3H]glucose (a measure of glycolysis) on Day 7 only. The results show that 10 microM DNP stimulates oxidative and glycolytic metabolism in Day-5 to Day-7 cattle embryos, but this does not fully explain the observed increase in developmental competence. We propose that partial inhibition or uncoupling of oxidative phosphorylation may reduce the level of intracellular reactive oxygen species production, thereby facilitating development.

A double-deletion mutation in the Pitx3 gene causes arrested lens development in aphakia mice.

The recessive aphakia (ak) mouse mutant is characterized by bilateral microphthalmia due to a failure of lens morphogenesis. We fine-mapped the ak locus to the interval between D19Umi1 and D19Mit9, developed new polymorphic markers, and mapped candidate genes by construction of a BAC contig. The Pitx3 gene, known to be expressed in lens primordia, shows zero recombination with the ak mutation on our intersubspecific intercross panel representing 1170 meioses. A recent report described a deletion in the intergenic region between Gbf1 and Pitx3 as the possible ak mutation. Our results differ in that we find not only the distant intergenic deletion, but also a much larger deletion directly in the Pitx3 gene, eliminating exon 1 and extending into intron 1 and the promoter region. Pitx3 transcript levels are severely reduced in ak/ak mice from E11.5 to newborn (5 +/- 1% of the wildtype levels at E13.5), while an involvement of the flanking Gbf1 and Cig30 genes in the aberrant lens development is highly unlikely based on expression analysis. We conclude that the ak mutation consists of two deletions, the larger of which removes part of Pitx3, indicating a crucial role of this gene in early lens development.

Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants.

Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.

Effect of different levels of intracellular cAMP on the in vitro maturation of cattle oocytes and their subsequent development following in vitro fertilization.

Serum, gonadotrophins, growth factors, and steroid hormones stimulate the in vitro maturation (IVM) of competent oocytes, acting, directly or indirectly, upon the adenylate cyclase pathway to produce the intracellular messenger, cAMP. The intracellular levels of cAMP in cattle cumulus-oocyte complexes (COC) were manipulated by adding to the collection and maturation media invasive adenylate cyclase (iAC), a toxin produced by the bacterium, Bordetella pertussis. High concentrations of iAC (1 or 5 microgram/ml) in the maturation medium inhibited the resumption of meiosis, while low concentrations (0.1 or 0.01 microgram/ml) resulted in high rates of maturation to the MII stage (92.6 +/- 2.5 and 98.5 +/- 1.4% respectively). The same low concentrations of iAC in the maturation medium resulted in rates of development to the blastocyst stage 8 days post insemination (30.1 +/- 4.2 and 45.1 +/- 3.9%, respectively), which were either not different, or significantly better, than those obtained after IVM in medium supplemented only with serum and gonadotrophins (36.1 +/- 2.9%). Finally, the addition of 0.1 microgram/ml iAC and 0.5 mM 3-isobutyl 1-methylxanthine (IBMX) in the collection medium significantly improved the blastocyst rate when IVM was performed in control medium or medium supplemented with 0.01 microgram/ml iAC (31.9 +/- 5.5 vs. 12.1 +/- 1.6 and 45.5 +/- 2.9 vs. 19.1 +/- 2.3% respectively). It is concluded that the maintenance of an optimal intracellular concentration of cAMP before and during IVM ensures a high developmental competence of bovine oocytes matured in medium without serum and hormones. Mol. Reprod. Dev. 54:86-91,1999.

Effect of time interval from insemination to first cleavage on the developmental characteristics, sex ratio and pregnancy rate after transfer of bovine embryos.

In vitro produced bovine zygotes show substantial variation in the time required to complete the first cell cycle and in their in vitro development potential. A number of reports have highlighted the fact that the fastest developing embryos in vitro are most likely to be comparable with their in vivo counterparts. At 24 h after IVF, presumptive zygotes were cultured in droplets of synthetic oviduct fluid medium. Droplets were examined at regular intervals and all cleaved embryos at each time point were transferred into new droplets and cultured separately for the duration of the experiment. All uncleaved zygotes were returned to the incubator and re-examined at the successive time points until 48 h after insemination, at which time the remaining uncleaved oocytes were retained as a group. A representative number of day 7 blastocysts from zygotes that had cleaved by 30 or 36 h were transferred to synchronized recipients and pregnancy was diagnosed by ultrasonography at day 35. Glucose and glutamine metabolism was examined in zygotes and blastocysts and compared retrospectively with time of first cleavage. A representative number of blastocysts from each of the cleavage groups was sexed using PCR. Data were analysed by chi-squared and regression analysis. Development to the blastocyst stage decreased as the time from insemination to first cleavage increased (r = 0.97, P < 0.03). There was no difference in blastocyst hatching, number of blastocyst cells or pregnancy rate between the 30 and 36 h groups. The overall sex ratio was 62% males (n = 258, P < 0.0001) and was not different in the 30 and 36 h groups (61%, n = 155 versus 63%, n = 95, respectively). These results indicate that although time of first cleavage has a major influence on the probability of an embryo developing to the blastocyst stage, once that stage is attained, subsequent developmental characteristics are unrelated to the time of first cleavage.

The effects of epidermal growth factor and insulin-like growth factor I on the metabolic activity, nuclear maturation and subsequent development of cattle oocytes in vitro.

The effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the maturation and subsequent development of cattle oocytes in vitro were evaluated in three experiments. Cumulus-oocyte complexes (COC) were collected from cattle ovaries and matured for 20-24 in control medium or in medium containing 50 ng EGF ml-1, 100 ng IGF-I ml-1, EGF + IGF-I, or 10% (v/V) fetal calf serum plus 0.1 i.u. human menopausal gonadotrophin ml-1 (hMG). In Expt 1, treatment with EGF + IGF-I stimulated cumulus expansion, the metabolism of pyruvate and glutamine, and nuclear maturation. In Expt 2, only the metabolic measurements from oocytes that reached metaphase II were considered, and EGF + IGF-I stimulated pyruvate metabolism to the same extent as serum + hMG. In Expt 3, the oocytes were fertilized after maturation culture, and the resultant embryos cultured for up to 8 days. The cleavage was greater in the EGF and EGF + IGF-I groups than in the controls but less than in the serum + hMG group. Moreover, the number of blastocyst cells at 7 days after insemination and the proportion of cleaved embryos that developed to the blastocyst stage by day 8 was greater in the serum + hMG group than in the control group indicating that maturation treatment can affect early embryonic development. In conclusion, EGF + IGF-I can stimulate cumulus expansion, oxidative metabolism, nuclear maturation and cleavage after fertilization of bovine oocytes in vitro. The relative effects of the treatments on oocyte pyruvate metabolism in Expts 1 and 2 generally paralleled their effects on cleavage and subsequent development in Expt 3, suggesting that mitochondrial function is related to developmental potential. Further investigation is required to determine which component(s) of serum or gonadotrophin treatment is responsible for the effects on subsequent embryonic development.

Effects of the in vitro chemical environment during early embryogenesis on subsequent development.

The development of the preimplantation embryo seems morphologically very simple, and embryologists previously assumed that an embryo that developed to the blastocyst stage was fully capable of normal development after transfer to the uterus of a recipient female. This complacency was disturbed by reports that exposure of early embryos to mutagens such as methylnitrosourea led to fetal abnormalities, decreased birth rates, and decreased life-span. Even more disturbing are recent reports that culture of early embryos in supposedly benign conditions can adversely affect their subsequent development. Techniques have been developed for the production of cattle and sheep embryos by in-vitro fertilization and by cloning. Such embryos must be cultured for several days before they can be transferred, and, in some cases, this has been related to abortion, very high birthweight, physical abnormalities and peri-natal mortality of the calves and lambs. This syndrome may result from an unbalanced development of the trophoblast relative to the inner-cell mass, possibly related to the presence of serum, glucose, or ammonium in the culture medium. An analogous phenomenon has been observed in human in-vitro fertilization where babies from single pregnancies have below-normal birth-weight. There is also evidence to suggest that the in-vitro environment of the gametes before fertilization can affect subsequent embryonal and fetal development. Exposure of mouse oocytes to vitrification solutions has been shown to lead to fetal malformations, and treatment of bull sperm with glutathione improves early embryo development. The common thread in these diverse observations is that development can be affected by events that occur long before any defect is apparent. Consequently, the production of a morphologically normal embryo is no guarantee that fetal development and post-natal life will be normal. This is of immediate concern in human reproductive medicine due to the increasing use of sperm injection for fertilization, and the emergence of in-vitro oocyte maturation. Further development and application of reproductive techniques would benefit from a toxicological evaluation of risk factors and exposure limits.

Batch analysis of the ATP content of bovine sperm, oocytes, and early embryos using a scintillation counter to measure the chemiluminescence produced by the luciferin-luciferase reaction.

This report describes an approach to using a commercial luciferin-luciferase kit and a scintillation counter for batch analysis of the ATP content of bovine sperm, oocytes and embryos. After ATP extraction, the luciferin-luciferase assay mixture was added to as many as 48 samples at one time, and the tubes counted once, and then again, in reverse order. The square-root of the geometric mean of the two counts for a tube was directly proportional to the ATP content. The measured ATP content was directly proportional to the number of sperm, oocytes, or blastocysts in a sample. The assay is sensitive enough to measure the ATP content of a single oocyte or blastocyst, or approximately 1000 sperm. The assay does not require the complicated and expensive automated sampling and mixing apparatus that is required for batch analysis using luminometer counting. This approach may significantly simplify the determination of the ATP content of large numbers of samples.

Structure/function studies on the pH-dependent actin-binding protein hisactophilin in Dictyostelium mutants.

Our previous studies have shown that the actin-binding protein hisactophilin from Dictyostelium discoideum is a candidate for organizing the actin cytoskeleton at the plasma membrane in a pH-dependent manner. To further characterize this interaction we isolated hisactophilin overexpression (hisII+) and hisactophilin minus (his-) mutants. D. discoideum contains two hisactophilin isoforms; both genes are independently transcribed and carry a short intron at the same position of the coding region. The deduced amino acid sequence of hisactophilin II showed a characteristic high content of 35 histidine residues out of a total 118 amino acids. After transformation of Dictyostelium AX2 wild-type cells with a genomic fragment designed to inactivate the hisactophilin I gene we obtained hisactophilin II overexpressing mutants (hisII+). Multiple integration of the vector led to strong overexpression of hisactophilin II which even outnumbered the actin concentration by a factor of two. Hisactophilin II protein showed the same biochemical properties as hisactophilin I during purification and in its pH-dependent binding to F-actin; as shown by mass spectrometry the hisactophilin II fraction was almost completely myristoylated despite of this high overexpression. The inactivation of both hisactophilin genes was achieved by gene replacement with a vector construct encompassing parts of gene I and gene II connected by a geneticin cassette. The properties of the hisII+ and his- cells with regard to growth in shaking culture and on Klebsiella plates, development, chemotaxis and morphology were not affected under normal conditions. However, the hisII+ transformants revealed a significant difference to wild-type cells and his- cells when the cytoplasmic pH was lowered by diethylstilbestrol (DES), a proton pump inhibitor. HisII+ cells were more resistant to the acidification; in contrast to AX2 wild-type cells and his- cells they did not form plasma membrane protrusions, showed an increase in F-actin content, and contained large clusters of F-actin. Lowering the internal pH caused an accumulation of hisactophilin below the plasma membrane. The fact that cells deficient in hisactophilin again lose resistance to acidification is in good agreement with the hypothesis that hisactophilin functions as a pH sensor at the plasma membrane by reversibly connecting the membrane with the actin cortical network upon local changes of the proton concentration.

Comparison of the effects of oviductal cell co-culture and oviductal cell-conditioned medium on the development and metabolic activity of cattle embryos.

The objective of this study was to compare the development and metabolic activity of cattle embryos co-cultured with bovine oviductal cells or cultured in serum-free medium previously conditioned by bovine oviductal cells. Zygotes were produced by in vitro fertilization of oocytes from bovine ovaries obtained from an abattoir. Development to the four-cell stage occurred by 48 h after fertilization in both culture systems, but co-cultured embryos reached the 16-cell stage by 96 h, whereas those cultured in conditioned medium did not do so until 24 h later. Similarly, the morula and blastocyst stages were reached 24 h earlier in co-culture than in conditioned medium. There were significantly more cells in the blastocysts from co-culture (96.8 +/- 6.1 versus 56.7 +/- 3.3; P < or = 0.0001). The metabolism of glutamine did not differ between embryos cultured in the two systems, but the metabolism of glucose was significantly greater in embryos cultured in conditioned medium. The first significant increase in glucose metabolism occurred between the four-cell and the 16-cell stages in embryos cultured in conditioned medium, but occurred between the 16-cell and morula stages in the co-cultured embryos, such that the glucose metabolism was significantly greater at the 16-cell stage in embryos cultured in conditioned medium compared with co-cultured embryos (6.5 +/- 1.0 versus 1.5 +/- 0.4 pmol per embryo per 3 h, P < or = 0.0001). The concentration of glucose was significantly less, and that of lactate significantly greater, in co-culture medium than in conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)