Development of pig embryos by nuclear transfer of cultured fibroblast cells

Pig fibroblast cells were transferred to enucleated oocytes by micromanipulation and electrofusion. The donor cells used for nuclear transfer were synchronized in presumptive G0 by serum starvation. In the first experiment, nuclear transfer was performed with fibroblasts that had either a smooth or a rough surface. A significant difference (p < 0.05) in the percentage of chromosome condensation (39.5%, 15/38 and 16.6%, 5/30) and nuclear formation (36.8%, 14/38 and 16.3%, 8/49) was found between the reconstructed embryos derived from the cells with smooth surface and with rough surfaces, respectively. The percentage of chromosome condensation (42.5%, 17/40 and 19.6%, 11/56) and nuclear formation (38.3%, 23/60 and 18.8%, 9/48) were higher (p < 0.05) in reconstructed embryos derived from small (15 microm) donor cells compared to large donor cells (20 microm), respectively. The percentage nuclei at 3 different time points (3, 6, and 9 hours in culture medium) was higher (p = 0.003) in the reconstructed embryos activated by thimerosal and dithiothreitol (20%, 36%, and 41.3%) compared to those without activation treatment (0%, 11.8%, and 22.2%). In addition, there was an increased percentage with nuclei as the time in culture increased from 3 to 9 hours (p = 0.029). The percentages of chromosome condensation (34.6%; 9/26) and nuclear formation (33.3%; 9/27) in nuclear transfer embryos were similar. The rate of blastocysts/morulae development (14.0%; 6/43) was low. However, 2 cavitated embryos (presumptive blastocysts) with 14 and 11 nuclei and 1 morula with 8 nuclei were obtained. This together with the above evidence indicate that the nuclei from pig fibroblast cells can be partially reprogrammed, which suggests that transfer of nuclei from fibroblast cells to in vitro matured oocytes resulting in production of identical or genetically altered pigs may be possible.     

Parthenogenetic activation of porcine oocytes after nuclear transfer

Mature porcine oocytes are arrested at metaphase II of meiosis. At fertilization, like all mammalian oocytes they exhibit a low frequency Ca(2+) oscillation lasting several hours. This oscillation is thought to be the signal that triggers resumption of meiosis and activates the developmental program of the oocyte. The signal transduction mechanism of the sperm-induced Ca(2+) signal is not known in detail, and attempts to generate the oscillation artificially have met with little success. Nevertheless, artificial activation of the oocyte is a crucial step during nuclear transfer. Methods are available to induce a transient elevation in the intracellular free Ca(2+) concentration to surpass the meiotic arrest and induce development of the constructed embryo. Further studies concentrating on the mechanism of Ca(2+) signaling during fertilization will help to improve the efficiency of the procedures used for parthenogenetic activation of the oocyte.     

The analgesic tropane analogue (+/-)-SM 21 has a high affinity for sigma2 receptors

The analgesic properties of the tropane analogue (+/-)-SM 21 have been attributed to the antagonism of presynaptic m2 receptors resulting in a potentiation of acetylcholine release. However, drugs targeting a number of other neurotransmitter receptors have been shown to enhance acetylcholine release. In the current study, in vitro studies were conducted in order to determine the affinity of (+/-)-SM 21 for serotonin 5-HT3, 5-HT4, and sigma receptors. Our results indicate that (+/-)-SM 21, and its structural congeners, have a relatively high affinity for sigma2 receptors relative to their reported affinity for muscarinic receptors. The higher affinity for sigma2 versus sigma1 receptors indicates that (+/-)-SM 21 may be a suitable lead compound for developing sigma2-selective ligands.     

Development of the techniques for nuclear transfer in pigs

Nuclear transfer in pigs was developed in the late 1980's. The techniques were based on previous studies in frogs, mice and cattle. Within stage nuclear transfer, pronuclear exchange, was followed by the transfer of nuclei from cleavage stage embryos. While these have resulted in term development, many problems remain. Recently progress on the problem of inadequate oocyte activation has been made and now there can be a refocus on the other aspects of the nuclear transfer procedure. The emphasis in developing the cloning/transgenic technology is easily justified, not so much by the ability to produce genetically identical animals for production agriculture, but for the potential to use a cell line that can be genetically engineered prior to the nuclear transfer. Pigs with specific genetic modifications will have a great impact on production agriculture as well as human medicine.     

Integral strategy for evaluation of fecal indicator performance in bird-influenced saline inland waters

Wild birds are an important nonpoint source of fecal contamination of surface waters, but their contribution to fecal pollution is mostly difficult to estimate. Thus, to evaluate the relation between feces production and input of fecal indicator bacteria (FIB) into aquatic environments by wild waterfowl, we introduced a new holistic approach for evaluating the performance of FIB in six shallow saline habitats. For this, we monitored bird abundance, fecal pellet production, and the abundance of FIB concomitantly with a set of environmental variables over a 9-month period. For estimating fecal pellet production, a new protocol of fecal pellet counting was introduced, which was called fecal taxation (FTX). We could show that, over the whole range of investigated habitats, bird abundance, FTX values, and FIB abundance were highly significantly correlated and could demonstrate the good applicability of the FTX as a meaningful surrogate parameter for recent bird abundances and fecal contamination by birds in shallow aquatic ecosystems. Presumptive enterococci (ENT) were an excellent surrogate parameter of recent fecal contamination in these saline environments for samples collected at biweekly to monthly sampling intervals while presumptive Escherichia coli and fecal coliforms (FC) were often undetectable. Significant negative correlations with salinity indicated that E. coli and FC survival was hampered by osmotic stress. Statistical analyses further revealed that fecal pollution-associated parameters represented one system component independent from other environmental variables and that, besides feces production, rainfall, total suspended solids (direct), and trophy (indirect) had significant positive effects on ENT concentrations. Our holistic approach of linking bird abundance, feces production, and FIB detection with environmental variables may serve as a powerful model for application to other aquatic ecosystems.     

Alternative states and positive feedbacks in restoration ecology

There is increasing interest in developing better predictive tools and a broader conceptual framework to guide the restoration of degraded land. Traditionally, restoration efforts have focused on re-establishing historical disturbance regimes or abiotic conditions, relying on successional processes to guide the recovery of biotic communities. However, strong feedbacks between biotic factors and the physical environment can alter the efficacy of these successional-based management efforts. Recent experimental work indicates that some degraded systems are resilient to traditional restoration efforts owing to constraints such as changes in landscape connectivity and organization, loss of native species pools, shifts in species dominance, trophic interactions and/or invasion by exotics, and concomitant effects on biogeochemical processes. Models of alternative ecosystem states that incorporate system thresholds and feedbacks are now being applied to the dynamics of recovery in degraded systems and are suggesting ways in which restoration can identify, prioritize and address these constraints.     

Rational design and synthesis of selective BACE-1 inhibitors

An effective approach for enhancing the selectivity of beta-site amyloid precursor protein cleaving enzyme (BACE 1) inhibitors is developed based on the unique features of the S1' pocket of the enzyme. A series of low molecular weight (<600) compounds were synthesized with different moieties at the P1' position. The selectivity of BACE 1 inhibitors versus cathepsin D and renin was enhanced 120-fold by replacing the hydrophobic propyl group with a hydrophilic propionic acid group.     

Ferment in the family tree: does a frugivorous dietary heritage influence contemporary patterns of human ethanol use?

Humans and apes are placed together in the superfamily Hominoidea. The evolutionary trajectory of hominoids is intimately bound up with the exploitation of ripe, fleshy fruits. Fermentation of fruit sugars by yeasts produces a number of alcohols, particularly ethanol. Because of their pre-human frugivorous dietary heritage, it has been hypothesized that humans may show pre-existing sensory biases associating ethanol with nutritional rewards. This factor, in turn, could influence contemporary patterns of human ethanol use. At present, there seems little evidence to support a view of selection specifically for ethanol detection or its utilization over the course of hominoid evolution. Ethanol concentration in wild fruits consumed by monkeys and apes is predicted to be low. Wild monkeys and apes avoid consumption of over-ripe fruits, the class showing notable ethanol concentrations, and for this reason, ethanol plumes may act as deterrents rather than attractants. Any energetic benefits to wild primates from ingested ethanol appear negligible, at best. Mice and rats show patterns of ethanol self-administration similar to humans, indicating that a frugivorous dietary heritage is not necessary for such behaviors. In the natural environment, ethanol is predicted to be just one of many alcohols, esters and related compounds routinely encountered by frugivorous primates and of no particular significance. The strong attraction ethanol holds for some individuals could be due to a broad range of genetic and environmental factors. In some humans, the appetite for ethanol appears related to the appetite for sugar. The predisposition some individuals display toward excessive ethanol consumption could involve features of their genetics and biochemical similarities of ethanol and carbohydrate. Regular low ethanol intake is hypothesized to lower the incidence of cardiovascular disease in humans, perhaps through its effects on body fat distribution. Such a benefit, if confirmed, would appear to relate to features of the contemporary human rather than pre-human diet.