DNA polymerase activity in trophoblast giant cells in the mouse /

In the developing mouse embryo, the diploid trophectoderm is known to undergo a diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the formation of giant nuclei. Studies of 13.5 day rat trophoblast derived from the parietal yolk sac have indicated a relatively low rate of DNA polymerase a activity, the noinnal eukaryotic replicase, in comparison to that of DNA polymerase g. These results have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase enzyme, DNA polymerase a. In order to determine whether a 'switch' from DNA polymerase to DNA polymerase is a necessary concomitant of the diploid to giant cell transformation, two distinct populations of trophoblast giant cells, the primary giant cell derived from the mural trophectoderm and the secondary giant cell derived from the polar trophoectoderm were used. These two populations of trophoblast giant cells can be obtained from the tissue outgrowths of 3.5da blastocysts and the extraembryonic ectoderm (EX) and ectoplacental cone (EPC) of 7.5 day embryos respectively. Tissue outgrowths were treated with aphidicolin, a specific reversible inhibitor of eukaryotic DNA polymerase a, on various days after explantation. The effect of aphidicolin treatment was assessed both qualitatively, using autoradiography and quantitatively by scintillation counting and Feulgen staining. 3 DNA synthesis was measured in control and treated cultures after a Hthymidine pulse. Scintillation counts of the embryo proper revealed that DNA synthesis was consistently inhibited by greater than 907. in the presence of aphidicolin. Inhibition of DNA synthesis in the EX and EPC varied between 81-957. and 82-987. respectively, indicating that most DNA synthesis was mediated by DNA polymerase a, but that a small but significant amount of residual synthesis was indicated. A qualitative approach was then applied to determine whether the apparent residual DNA synthesis was restricted to a subpopulation of giant cells or whether all giant cells displayed a low level of DNA synthesis. Autoradiographs of the ICM of blastocysts and the embryo proper of 7.5da embryos, which acted as diploid control population, was completely inhibited regardless of duration in explant culture. In contrast, primary trophoblast giant cells derived from blastocysts and secondary giant cells derived from the EX and EPC were observed to possess some heavily labelled cells after aphidicolin treatment. These results suggest that although DNA polymerase a is the primary replicating enzyme responsible for endoreduplication in mouse trophoblast giant cells, some nonactivity is also observed. A DNA polymerase assay employing tissue lysates of outgrown 7.5da embryo, EX and EPC tissues was used to attempt to confirm the presence of higher nonactivity in tissues possessing trophoblast giant cells. Employing a series of inhibitors of DNA polymerases, it would appear that DNA polymerase a is the major polymerase active in all tissues of the 7.5da mouse embryo. The nature of the putative residual DNA synthetic activity could not be unequivically determined in this study. Therefore, these results suggest that both primary and secondary trophoblast giant cells possess and use DNA polymerase a in endoreduplicative DNA synthesis. It would appear that the high levels of DNA polymerase g activity reported in trophoblast tissue derived from the 13.5 da rat yolk sac was not a general feature of all endoreduplication.

Activity of brainstem cholinergic neurons during 22 kHz ultrasonic vocalization in rats /

Adult rats emit 22 kHz ultrasonic alann calls in aversive situations. This type of call IS a component of defensive behaviour and it functions predominantly to warn conspecifics about predators. Production of these calls is dependent on the central cholinergic system. The laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) contain largely cholinergic neurons, which create a continuous column in the brainstem. The LDT projects to structures in the forebrain, and it has been implicated in the initiation of 22 kHz alarm calls. It was hypothesized that release of acetylcholine from the ascending LDT terminals in mesencephalic and diencephalic areas initiates 22 kHz alarm vocalization. Therefore, the tegmental cholinergic neurons should be more active during emission of alarm calls. The aim of this study was to demonstrate increased activity of LDT cholinergic neurons during emission of 22 kHz calls induced by air puff stimuli. Immunohistochemical staining of the enzyme choline acetyltransferase identified cell bodies of cholinergic neurons, and c-Fos immunolabeling identified active cells. Double labeled cells were regarded as active cholinergic cells. There were significantly more (p

Broad reduction and adoption in ring-billed gulls (Larus Delaworensis): the potential for inergenerational conflict

Patterns of intra-clutch egg size variation and intra-clutch hatch intervals in the Ring-billed gull (Larus delawarensis) were documented during the peak nesting period of two consecutive breeding seasons, at a colony near Port Colborne, Ontario. Egg size decreased with laying order; third laid eggs were significantly smaller than first laid eggs. Hatching of the third egg was delayed from that of first and second eggs. Intraclutch egg size differences established initial size disparities among chicks at hatch. Hatch intervals further exaggerated size disparities during the early post brood completion period. Competitive asymmetries among chicks were associated with increased mortality rates among third hatched chicks despite the lack of evidence of a sibling feeding hierarchy. Fledging success in 1987 was greater than in 1988. A "brood reduction strategy" appears to have enabled parents in 1987, to obtain an extra unit of reproductive fitness, while in 1988 parents were often unable to raise the entire brood and third chicks likely represented insurance reproductive value. Experimental broods (1988) were created in which hatch intervals were double those of natural intervals. The size disparities among chicks were significantly greater than in control broods, and the pattern of mortality among chicks suggested that first chicks benefited at a cost to second and third chicks. Parents of peak experimental broods achieved a fledging success rate similar to that of control broods. Characteristics of chick adoptions were also recorded. In each study year, 9 chicks abandoned their natal territories, 6 of which were adopted. Chicks consistently established themselves into broods where they were older than resident chicks. No direct evidence of cost to foster parents, or benefits to adopted chicks was obtained, although fledging success of adopted chicks was high.

METHOXYPYRAZINES AND LADYBUG TAINT IN WINES

Methoxypyrazines are aroma active compounds found in many wine varietals. These compounds can be of either grape-derived nature or can be introduced into wines via Coccinellidae beetles. Regardless of their origin, methoxypyrazines can have either a beneficial role for wine quality, contributing to the specificity of certain wine varietals (Cabernet sauvignon, Cabernet franc, Sauvignon blanc) or a detrimental role, particularly at higher concentrations, resulting in overpowering green, unripe and herbaceous notes. When methoxypyrazines of exogenous nature are responsible for these unpleasant characteristics, wines are considered to be affected by what is generally known as Ladybug taint (LBT). This is work is a collection of studies seeking to create a sensitive analytical method for the detection and quantification of methoxypyrazines in wines; to investigate the role of different Coccinellidae species in the tainting of wines with LBT and identify the main compounds in ladybug tainted wines responsible for the typical green herbaceous characteristics; to determine the human detection threshold of 2,5-dimethyl-3-methoxypyrazine in wines as well as investigate its contribution to the aroma of wines; and finally to survey methoxypyrazine concentrations in a large set of wines from around the world. In the first study, an analytical method for the detection and quantitation of methoxypyrazines in wines was created and validated. The method employs multidimensional Gas Chromatography coupled with Mass Spectrometry to detect four different methoxypyrazines (2,5-dimethyl-3-methoxypyrazine, isobutyl methoxypyrazine, secbutyl methoxypyrazine and isopropyl methoxypyrazines) in wine. The low limits of detection for the compounds of interest, improved separation and isolation capabilities, good validation data, as well as the ease of use recommend this method as a good alternative to the existing analytical methods for methoxypyrazine detection in wine. In the second study the capacity of two Coccinellidae species, found in many wine regions – Harmonia axyridis and Coccinella septempunctata - to taint wines is evaluated. Coccinella septempunctata is shown to be as capable as causing LBT in wines as Harmonia axyridis. Dimethyl methoxypyrazine, previously thought to be of exogenous nature only (from Coccinellidae haemolymph), is also detected in control (untainted) wines. The main odor active compounds in LBT wines are investigated through Aroma Extract Dilution Assay. These compounds are identified as isopropyl methoxypyrazine, sec- and iso- butyl methoxypyrazine. In the third study, the human detection threshold for dimethyl methoxypyrazine in wine is established to be 31 ng/L in the orthonasal modality and 70 ng/L retronasally. After wines spiked with various amounts of dimethyl methoxypyrazine are evaluated sensorally, dimethyl methoxypyrazine causes significant detrimental effects to wine aroma at a concentration of 120 ng/L. The final study examines methoxypyrazine (dimethyl methoxypyrazine, isopropyl methoxypyrazine, secbutyl methoxypyrazine and isobutyl methoxypyrazine) concentrations in 187 wines from around the world. Dimethyl methoxypyrazine is detected in the majority of the red wines tested. Data are interpreted through statistical analyses. A new measure for predicting greenness/herbaceousness in wines - methoxypyrazine “total impact factor” is proposed.

Mode of action of a Drosophila FMRFamide in inducing muscle contraction

Drosophila melanogaster is a model system for examining the mechanisms of action of neuropeptides. DPKQDFMRFamide was previously shown to induce contractions in Drosophila body wall muscle fibres in a Ca(2+)-dependent manner. The present study examined the possible involvement of a G-protein-coupled receptor and second messengers in mediating this myotropic effect after removal of the central nervous system. DPKQDFMRFamide-induced contractions were reduced by 70% and 90%, respectively, in larvae with reduced expression of the Drosophila Fmrf receptor (FR) either ubiquitously or specifically in muscle tissue, compared with the response in control larvae in which expression was not manipulated. No such effect occurred in larvae with reduced expression of this gene only in neurons. The myogenic effects of DPKQDFMRFamide do not appear to be mediated through either of the two Drosophila myosuppressin receptors (DmsR-1 and DmsR-2). DPKQDFMRFamide-induced contractions were not reduced in Ala1 transgenic flies lacking activity of calcium/calmodulin-dependent protein kinase (CamKII), and were not affected by the CaMKII inhibitor KN-93. Peptide-induced contractions in the mutants of the phospholipase C-β (PLCβ) gene (norpA larvae) and in IP3 receptor mutants were similar to contractions elicited in control larvae. The peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. Peptide-induced contractions were not potentiated by 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and were not antagonized by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. Additionally, exogenous application of arachidonic acid failed to induce myogenic contractions. Thus, DPKQDFMRFamide induces contractions via a G-protein coupled FMRFamide receptor in muscle cells but does not appear to act via cAMP, cGMP, IP3, PLC, CaMKII or arachidonic acid.

Frontal Lobe Function and Performance Monitoring following Total Sleep Deprivation

Imaging studies have shown reduced frontal lobe resources following total sleep deprivation (TSD). The anterior cingulate cortex (ACC) in the frontal region plays a role in performance monitoring and cognitive control; both error detection and response inhibition are impaired following sleep loss. Event-related potentials (ERPs) are an electrophysiological tool used to index the brain's response to stimuli and information processing. In the Flanker task, the error-related negativity (ERN) and error positivity (Pe) ERPs are elicited after erroneous button presses. In a Go/NoGo task, NoGo-N2 and NoGo-P3 ERPs are elicited during high conflict stimulus processing. Research investigating the impact of sleep loss on ERPs during performance monitoring is equivocal, possibly due to task differences, sample size differences and varying degrees of sleep loss. Based on the effects of sleep loss on frontal function and prior research, it was expected that the sleep deprivation group would have lower accuracy, slower reaction time and impaired remediation on performance monitoring tasks, along with attenuated and delayed stimulus- and response-locked ERPs. In the current study, 49 young adults (24 male) were screened to be healthy good sleepers and then randomly assigned to a sleep deprived (n = 24) or rested control (n = 25) group. Participants slept in the laboratory on a baseline night, followed by a second night of sleep or wake. Flanker and Go/NoGo tasks were administered in a battery at 1O:30am (i.e., 27 hours awake for the sleep deprivation group) to measure performance monitoring. On the Flanker task, the sleep deprivation group was significantly slower than controls (p's <.05), but groups did not differ on accuracy. No group differences were observed in post-error slowing, but a trend was observed for less remedial accuracy in the sleep deprived group compared to controls (p = .09), suggesting impairment in the ability to take remedial action following TSD. Delayed P300s were observed in the sleep deprived group on congruent and incongruent Flanker trials combined (p = .001). On the Go/NoGo task, the hit rate (i.e., Go accuracy) was significantly lower in the sleep deprived group compared to controls (p <.001), but no differences were found on false alarm rates (i.e., NoGo Accuracy). For the sleep deprived group, the Go-P3 was significantly smaller (p = .045) and there was a trend for a smaller NoGo-N2 compared to controls (p = .08). The ERN amplitude was reduced in the TSD group compared to controls in both the Flanker and Go/NoGo tasks. Error rate was significantly correlated with the amplitude of response-locked ERNs in control (r = -.55, p=.005) and sleep deprived groups (r = -.46, p = .021); error rate was also correlated with Pe amplitude in controls (r = .46, p=.022) and a trend was found in the sleep deprived participants (r = .39, p =. 052). An exploratory analysis showed significantly larger Pe mean amplitudes (p = .025) in the sleep deprived group compared to controls for participants who made more than 40+ errors on the Flanker task. Altered stimulus processing as indexed by delayed P3 latency during the Flanker task and smaller amplitude Go-P3s during the Go/NoGo task indicate impairment in stimulus evaluation and / or context updating during frontal lobe tasks. ERN and NoGoN2 reductions in the sleep deprived group confirm impairments in the monitoring system. These data add to a body of evidence showing that the frontal brain region is particularly vulnerable to sleep loss. Understanding the neural basis of these deficits in performance monitoring abilities is particularly important for our increasingly sleep deprived society and for safety and productivity in situations like driving and sustained operations.