Production of chimeric rabbits from morulae by a simple procedure

Experiments were conducted to develop a simple and reliable technique to produce chimeric rabbits from morula stage embryos. In Experiments 1 and 2, an in-vitro test of viability was initially performed by culturing embryos to the blastocyst stage. Ninety-three percent of the “chimeric” embryos developed to the blastocyst stage compared to 94% for controls when embryos were manipulated soon after collection (Exp. 1). Eighty-one percent chimeric embryos and 78% control embryos developed to blastocyst stage when embryos were held at room temperature for 4 hr (Exp. 2). In Experiment 3, enough morula-stage embryos were available from true breeding Dutch-belted and albino rabbits to form potentially 67 diverse “color” pairs. These micromanipulated pairs of morulae were successfully combined to produce 64 chimeric embryos (96%, 64/67). They were transferred to the uteri of seven recipient does and three became pregnant producing 13 young. Four of the young exhibited substantial overt chimerism (31%) and one more was a possible chimera.     

Substrate specificity of aspartate transcarbamylase. Interaction of the enzyme with analogs of aspartate and succinate

The ability of aspartate transcarbamylase from Escherichia coli to catalyze carbamylation of amino acids other than the natural substrate, L-aspartate, was examined. Cysteine, cysteate, cysteinesulfinate, and 3-nitroalanine showed kcat values at pH 7 of 0.16, 0.58, 5.2, and 62 s-1, respectively, while kcat with aspartate was 320 s-1. In a parallel study, competitive inhibition constants of 3-nitropropionate, 3-mercaptopropionate, 3-sulfopropionate, and 3-sulfinopropionate were found to be high, about 0.1 M, compared with that of succinate, 0.56 mM. Although cysteinesulfinate had low activity as a substrate, the pH dependences of kcat and kcat/Km in H2O and D2O observed with the compound closely paralleled those of aspartate. The results of these studies suggest that substrate specificity and reactivity are achieved in part by a strong, highly specific interaction of one or more active site residues with the beta-carboxylate of L-aspartate. Unlike the sigmoidal kinetics found with aspartate, saturation of native aspartate transcarbamylase by cysteine sulfinate showed a lack of cooperativity, even under conditions of activation of the reaction by ATP and inhibition by CTP. The cysteinesulfinate reaction was increased 9-fold by the bisubstrate analog N-phosphonacetyl-L-aspartate. These results were interpreted in terms of an inability of cysteinesulfinate to cause the allosteric conformational change promoted by aspartate.     

Decoding the ‘zeep’ complex: quantitative analysis of interspecific variation in the nocturnal flight calls of nine wood warbler species (Parulidae spp.)

ABSTRACT

The “zeep” complex consists of nine birds that produce nocturnal flight calls with similar acoustic features. Our inability to distinguish these calls inhibits the acoustic monitoring of these species. We test the hypothesis that flight calls of nine warblers in the “zeep” complex show sufficient acoustic differences to allow differentiation. We investigate divergence in these vocalizations by recording birds held for banding and collecting additional recordings from sound libraries. We used three approaches to compare calls between species: analysis of variance in acoustic properties, discriminant analysis of acoustic properties, and spectrographic cross-correlation. The first approach revealed five species that were different in one or more acoustic properties. The second approach revealed a level of assignment to the correct species (73%) that exceeded levels expected by chance (36%). The third approach revealed calls of seven species to be significantly more similar to conspecific calls than heterospecific calls. Our results suggest the calls of many members of the “zeep” complex exhibit species-specific differences in structure, which may allow differentiation of at least five “zeep” species based on call alone. We advocate for the combined use of these three approaches for the comparison of “zeep” calls in future flight call studies.     

Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae

Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2,38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate (1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, (2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and (3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts. © 1993 Wiley-Liss, Inc.     

Acute and Chronic Mood and Apathy Outcomes from a Randomized Study of Unilateral STN and GPi DBS

Objective

To study mood and behavioral effects of unilateral and staged bilateral subthalamic nucleus (STN) and globus pallidus internus (GPi) deep brain stimulation (DBS) for Parkinson''s disease (PD).

Background

There are numerous reports of mood changes following DBS, however, most have focused on bilateral simultaneous STN implants with rapid and aggressive post-operative medication reduction.

Methods

A standardized evaluation was applied to a subset of patients undergoing STN and GPi DBS and who were also enrolled in the NIH COMPARE study. The Unified Parkinson Disease Rating Scale (UPDRS III), the Hamilton depression (HAM-D) and anxiety rating scales (HAM-A), the Yale-Brown obsessive-compulsive rating scale (YBOCS), the Apathy Scale (AS), and the Young mania rating scale (YMRS) were used. The scales were repeated at acute and chronic intervals. A post-operative strategy of non-aggressive medication reduction was employed.

Results

Thirty patients were randomized and underwent unilateral DBS (16 STN, 14 GPi). There were no baseline differences. The GPi group had a higher mean dopaminergic dosage at 1-year, however the between group difference in changes from baseline to 1-year was not significant. There were no differences between groups in mood and motor outcomes. When combining STN and GPi groups, the HAM-A scores worsened at 2-months, 4-months, 6-months and 1-year when compared with baseline; the HAM-D and YMRS scores worsened at 4-months, 6-months and 1-year; and the UPDRS Motor scores improved at 4-months and 1-year. Psychiatric diagnoses (DSM-IV) did not change. No between group differences were observed in the cohort of bilateral cases.

Conclusions

There were few changes in mood and behavior with STN or GPi DBS. The approach of staging STN or GPi DBS without aggressive medication reduction could be a viable option for managing PD surgical candidates. A study of bilateral DBS and of medication reduction will be required to better understand risks and benefits of a bilateral approach.     

Post-contest behaviour in black-capped chickadees (<Emphasis Type="Italic">Poecile atricapillus</Emphasis>): loser displays,not victory displays,follow asymmetrical countersinging exchanges

Victory displays are behaviours that occur after the conclusion of a signaling contest, performed solely by the contest winner. Victory displays may reinforce the dominance of the winner either to the loser or to other conspecifics within signaling range. Victory displays are poorly studied despite the significant consequences that post-conflict behaviour may have on the individuals involved. We examined the period immediately following 50 territorial countersinging contests between males in 10 neighbourhoods of black-capped chickadees (Poecile atricapillus) of known dominance rank. We characterized the post-contest singing behaviour of chickadees and evaluated whether post-contest behaviour is consistent with victory displays. Using a 16-microphone acoustic location system to simultaneously record entire neighbourhoods of breeding chickadees, we isolated 50 dyadic countersinging contests and measured the vocal behaviour of the contestants in the minutes following each interaction. Eighty-six percent of contests were followed by a period of solo singing by one of the contestants, while 14% were followed by silence. The post-contest singer was most often the contestant who held a subordinate dominance position in the previous winter’s dominance hierarchy; dominant males performed post-contest song bouts significantly less often. Asymmetry in overlapping between contestants did not predict which bird sang a post-contest bout. However, in a significant majority of cases, the post-contest singer was pitch-matched by his opponent during the contest more than he pitch-matched his opponent. Our results indicate that male chickadees do not perform acoustic victory displays after countersinging contests. In contrast, the post-contest behaviour of territorial chickadees is more consistent with a “loser display”.     

The Ene Reaction of Singlet Oxygen with Olefins

The reactions of singlet oxygen (¹O₂) with cis and trans butenes-1,1,1-d₃, at—80°C in Freon-11, show a product isotope effect (k_H/k_D) of 1.38 and 1.25 respectively. Isomerization of the starting materials or formation of dioxetanes were not observed during the course of the photooxygenation. Together with the isotope effects on the reactions of tetramethylethylene-d₆ isomers with singlet oxygen, these results require the reversible formation of a perepoxide or charge transfer intermediate.     

Comparison of six-day bovine embryo development in uterine tube (oviduct) epithelial cell co-culture versus in vivo development in the cow

A study was designed to evaluate and compare the appearance of embryos recovered from donor cows on Day 6 to embryos from in vivo fertilized cow zygotes developed to Day 6 on uterine tube (oviduct) epithelial cell co-culture using serum-free CZB medium. Embryo stage of development and quality score were assessed. Hoechst 33342 DNA stain was then used to determine the total number of blastomeres, the number of poor nuclei and the number of nuclei in mitosis. Mean cell counts did not differ for the 70 embryos evaluated in each group (65 cells in vivo, 61 cells in vitro). The percentage of transferable emryos (excellent, good or fair quality), in each group also did not differ (57% in vivo, 56% in vitro). There were no significant differences in any of the measured parameters. Our findings suggest that co-culture of in vivo produced cow zygotes can result in embryos comparable in developmental stage and quality to embryos developed in vivo in the cow for 6 d.     

NuMA localization,stability, and function in spindle orientation involve 4.1 and Cdk1 interactions

The epidermis is a multilayered epithelium that requires asymmetric divisions for stratification. A conserved cortical protein complex, including LGN, nuclear mitotic apparatus (NuMA), and dynein/dynactin, plays a key role in establishing proper spindle orientation during asymmetric divisions. The requirements for the cortical recruitment of these proteins, however, remain unclear. In this work, we show that NuMA is required to recruit dynactin to the cell cortex of keratinocytes. NuMA''s cortical recruitment requires LGN; however, LGN interactions are not sufficient for this localization. Using fluorescence recovery after photobleaching, we find that the 4.1-binding domain of NuMA is important for stabilizing its interaction with the cell cortex. This is functionally important, as loss of 4.1/NuMA interaction results in spindle orientation defects, using two distinct assays. Furthermore, we observe an increase in cortical NuMA localization as cells enter anaphase. Inhibition of Cdk1 or mutation of a single residue in NuMA mimics this effect. NuMA''s anaphase localization is independent of LGN and 4.1 interactions, revealing two distinct mechanisms responsible for NuMA cortical recruitment at different stages of mitosis. This work highlights the complexity of NuMA localization and reveals the importance of NuMA cortical stability for productive force generation during spindle orientation.     

In vitro capacitation and fertilizing ability of ejaculated rabbit sperm treated with lysophosphatidylcholine

Four experiments were replicated 1) to establish dose-response relationships between lysophosphatidylcholine (LPC), sperm motility, and the acrosome reaction (AR), 2) to evaluate the influence of rabbit serum (RS) on these endpoints, 3) to compare buck differences in induction of the AR, and 4) to examine fertilizing ability in vitro of sperm tested under the first three objectives. Semen was collected from Dutch-belted rabbits, washed once by centrifugation, resuspended, and preincubated for 2 or 4 hr in a chemically defined medium (DM), DM plus 20% RS, or BSA-free DM plus 20% RS at 37°C. At the end of preincubation LPC was added to the preincubated sperm at concentrations of from 0 to 100 μg/ml. Sperm were examined .5–4 hr later for AR and sperm motility. For in vitro fertilization, sperm and ova were coincubated in DM up to 24 hr after insemination and in a more complex medium for another 24 hr. Addition of LPC to 4-hr-preincubated sperm was more effective for inducing the AR than addition to 2-hr-preincubated sperm. A significant increase (P < .05) in the AR occurred in 15 and 30 min following exposure to 100 and 80 μg of LPC per ml, respectively, but the higher concentration of LPC decreased sperm motility. Addition of 20% RS to DM with or without BSA surprisingly inhibited the AR but maintained sperm motility, as expected. Bucks differed (P < .05) in the initial percentage and the induced percentage of AR sperm. For the AR the optimal concentration of LPC per ml was 80 μg, but for in vitro fertilization 60 μg tended to be superior.