New developments reproductive technologies in deer

In vitro embryo production is the platform for advanced reproductive technologies, such as cloning. The in vitro embryo production system developed for farmed red deer (Cervus elaphus) evolved along similar lines to that pioneered by other domestic species researchers. However, applying existing in vitro embryo production methods from these other species resulted in limited success and has necessitated developing a species-specific methodology for red deer based on the their physiology. Analysis of oviduct fluid led to the development of a semi-defined fertilization and culture media system, Deer Synthetic Oviduct Fluid (DSOF), which resulted in successful culture of red deer embryos to the blastocyst stage. Transvaginal ultrasound-guided ovarian examination and ovum pickup has enabled the study of seasonality constraint and propagation from selected female genetics, respectively. During the 4-month breeding season (April-July), 15% of cleaved oocytes developed to blastocysts, whereas no blastocysts developed from oocytes collected after July. The process of developing an in vitro embryo production system for farmed red deer may serve as a beneficial model for the propagation of endangered cervine species.     

Enhancement of therapeutic protein in vivo activities through glycoengineering

Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).     

Spatial trends in the sighting dates of British butterflies

A strong relationship between appearance dates and temperature has been demonstrated over two decades for most British butterflies. Given this relationship over time, this paper tests whether comparable spatial trends in timing are also apparent. A major survey of British butterflies is used to calculate mean sighting dates of adults across the country, and these are compared with geographic patterns in temperature. With the use of regression techniques, we calculated latitudinal (south-north) and longitudinal (east-west) gradients in sighting date and temperature. The majority of butterflies appear later in the east of Britain where temperatures are lower during summer, but not the rest of the year. Most butterflies are also seen later in the cooler north of the country, by upto 3-4 days/100 km. However, no geographical relationship between temperature and timing of appearance was detected for over a third of the species analysed, suggesting their populations may be adapted to their local climates. We suggest possible mechanisms for this and discuss the implications of such adaptation for the ability of butterfly species to respond to rapid climate warming.     

Mechanism of glycogen supercompensation in rat skeletal muscle cultures

A model to study glycogen supercompensation (the significant increase in glycogen content above basal level) in primary rat skeletal muscle culture was established. Glycogen was completely depleted in differentiated myotubes by 2 h of electrical stimulation or exposure to hypoxia during incubation in medium devoid of glucose. Thereafter, cells were incubated in medium containing glucose, and glycogen supercompensation was clearly observed in treated myotubes after 72 h. Peak glycogen levels were obtained after 120 h, averaging 2.5 and 4 fold above control values in the stimulated- and hypoxia-treated cells, respectively. Glycogen synthase activity increased and phosphorylase activity decreased continuously during 120 h of recovery in the treated cells. Rates of 2-deoxyglucose uptake were significantly elevated in the treated cells at 96 and 120 h, averaging 1.4–2 fold above control values. Glycogenin content increased slightly in the treated cells after 48 h (1.2 fold vs. control) and then increased considerably, achieving peak values after 120 h (2 fold vs. control). The results demonstrate two phases of glycogen supercompensation: the first phase depends primarily on activation of glycogen synthase and inactivation of phosphorylase; the second phase includes increases in glucose uptake and glycogenin level.     

Glycogen metabolism in rat heart muscle cultures after hypoxia

Elevated glycogen levels in heart have been shown to have cardioprotective effects against ischemic injury. We have therefore established a model for elevating glycogen content in primary rat cardiac cells grown in culture and examined potential mechanisms for the elevation (glycogen supercompensation). Glycogen was depleted by exposing the cells to hypoxia for 2 h in the absence of glucose in the medium. This was followed by incubating the cells with 28 mM glucose in normoxia for up to 120 h. Hypoxia decreased glycogen content to about 15% of control, oxygenated cells. This was followed by a continuous increase in glycogen in the hypoxia treated cells during the 120 h recovery period in normoxia. By 48 h after termination of hypoxia, the glycogen content had returned to baseline levels and by 120 h glycogen was about 150% of control. The increase in glycogen at 120 h was associated with comparable relative increases in glucose uptake (~ 180% of control) and the protein level of the glut-1 transporter (~ 170% of control), whereas the protein level of the glut-4 transporter was decreased to < 10% of control. By 120 h, the hypoxia-treated cells also exhibited marked increases in the total (~ 170% of control) and fractional activity of glycogen synthase (control, ~ 15%; hypoxia-treated, ~ 30%). Concomitantly, the hypoxia-treated cells also exhibited marked decreases in the total (~ 50% of control) and fractional activity of glycogen phosphorylase (control, ~ 50%; hypoxia-treated, - 25%). Thus, we have established a model of glycogen supercompensation in cultures of cardiac cells that is explained by concerted increases in glucose uptake and glycogen synthase activity and decreases in phosphorylase activity. This model should prove useful in studying the cardioprotective effects of glycogen.     

Fluorescence polarization: a novel indicator of cardiomyocyte contraction

The changes measured in intracellular fluorescein fluorescence polarization (IFFP) are used as a new tool for tracing cytoplasmic effects during contractile cycles of cardiac myocytes (1-2-day-old rat hearts), in addition to the established Ca(2+) monitoring and/or videometric methods of tracking cell-shortening. This novel method was found to be non-intrusive to the contraction cycles. The decay of the transient IFFP signal (from 0.220+/-0.01 to 0.170+/-0.013) seems to be closely related to the extended phase of contractile activation. This fact was further supported when Ca(2+) exchanger inhibitor was introduced and significantly decreased (90%) the rate of beats of contraction and IFFP, but not the Ca(2+) beat rate changes. This result suggests that the IFFP indicator is probably associated with the physiological activation, rather than with Ca(2+) alterations. The IFFP measure monitors the average of effective changes in the micro-viscosity of the cytoplasm protein matrix, associated with cellular activation.     

The kin composition of social groups: trading group size for degree of altruism

Why some social systems form groups composed of kin, while others do not, has gone largely untreated in the literature. Using an individual-based simulation model, we explore the demographic consequences of making kinship a criterion in group formation. We find that systems where social groups consist of one-generation breeding associations may face a serious trade-off between degree of altruism and group size that is largely mediated by their kin composition. On the one hand, restricting groups to close kin allows the evolution of highly altruistic behaviors but may limit group size to suboptimal levels, the more severely so the smaller the intrinsic fecundity of the species and the stricter the kin admission rule. Group size requirements, on the other hand, can be met by admitting nonkin into groups, but not without limiting the degree of altruism that can evolve. As a solution to this conundrum, we show that if helping roles within groups are assigned through a lottery rather than being genetically determined, maximum degrees of altruism can evolve in groups of nonrelatives of any size. Such a "lottery" mechanism may explain reproductive and helping patterns in organisms as varied as the cellular slime molds, pleometrotic ants, and Galapagos hawks.     

External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba(2+) binding kinetics and the concentration and voltage dependence of Ba(2+) steady-state block. Our results indicate that extracellular Ba(2+) exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba(2+) site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba(2+) site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba(2+) on channel gating in low external K(+) solutions. Ba(2+) binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K(+) attenuates Ba(2+) inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K(+) channels, KCNQ1 channels display significant structural and functional uniqueness.     

Maternal autoimmune diseases and immunologically induced embryonic and fetoplacental damage

BACKGROUND: Autoimmune diseases are a group of illnesses in which autoantibodies are produced against various organs, presenting with a variety of clinical symptoms. In this review, we discuss the different aspects of autoimmune diseases in pregnancy. We also describe experimental models that help to understand the etiology and pathogenesis of the effects of this maternal disease on the developing embryo, fetus and placenta. METHODS: The possible direct effects of sera or IgG obtained from women with systemic lupus erythematosus/antiphospholipid syndrome (SLE/APS) and recurrent pregnancy loss (RPL) were examined on cultured 10.5- and 11.5-day-old rat embryos and on cultured human placental explants, as compared to sera from healthy women or synthetic medium that were used as controls. In addition, we examined the effects of the sera obtained from these women after successful treatment that allowed the birth of normal infants. RESULTS: We observed increased embryonic death and anomalies in embryos cultured on sera and IgG from SLE/APS women. Similarly, when human placental explants were cultured on these sera, trophoblastic cell growth was reduced and apoptotic rate was increased. Successful treatment also reduced the damage caused by the sera from these women in the cultured embryos and placentas. CONCLUSIONS: Our results, and the cited studies, point to the important role of the placental damage in the etiology of RPL associated with SLE/APS. Animal models, both in vivo and in vitro, as well as cultured early human placental explants can be used successfully to understand some of the pathogenic aspects of SLE/APS and RPL.