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.     

Changes in body mass and organ size during remigial moult in common scoter Melanitta nigra

The “cost‐benefit” hypothesis states that avian body organs show mass changes consistent with the trade‐off between their functional importance and maintenance cost, which may vary throughout the annual cycle. Flightless moulting common scoter Melanitta nigra in Danish marine waters select rich undisturbed offshore feeding areas lacking predators, suggesting active feeding during moult. We tested four predictions relating to organ size during flightlessness in moulting male common scoter under this hypothesis. Namely that (i) pectoral muscles would show atrophy followed by hypertrophy, but that there would be no change in (ii) leg muscles and heart (the locomotory architecture required to sustain diving for food), (iii) digestive organs and liver (required to process food), or (iv) fat deposits (because birds could fulfil daily energy requirements from locally abundant food resources). Dissection of scoters collected at different stages during wing moult south of the Danish island of Læsø provided data on organ size that were consistent with these predictions. Pectoral muscle mass showed a c.23% atrophy during the middle of the flightless period relative to that at the end of moult. There was no significant loss in leg muscle, heart, digestive organs (except gizzard mass), liver, fat reserves or body mass with remigial growth. These findings are consistent with the hypothesis that common scoter moult in a rich feeding area, and rely on their diet to meet the nutritional requirements of remigial moult. These results differ in detail from those of a similar study of terrestrial feeding moulting greylag geese Anser anser, but because of the widely differing ecology of the species concerned, both sets of findings provide strong support for the hypothesis that variations in phenotypic plasticity in size of fat stores, locomotor and digestive organs can be interpreted as evolutionary adaptations to meet the conflicting needs (feather growth, nutritional challenges and predator avoidance) of the flightless moult period in different Anatidae species.     

Moving as a reason for pet relinquishment: a closer look

One of the most frequently given reasons for relinquishing a companion animal to an animal shelter is that the person or family is moving. Telephone interviews conducted with 57 caregivers who relinquished animal companions to a shelter in the midwestern United States covered details of the move, characteristics both of the caregivers and the animals, and efforts to avoid relinquishment. A human-nonhuman animal bonding scale also was administered. Although some participants had additional reasons for relinquishment, the majority had given up their pets solely because they were moving. Most had relatively low income, were moving for employment reasons, and were renting their homes. Landlord restrictions were an important factor in relinquishment. High scores on the bonding scale and spontaneous expressions of discomfort and sorrow suggest that external pressures overrode attachment to the animal and the pain of relinquishment.     

Effects of nitrate and acetate availability on chloroform production during carbon tetrachloride destruction

Fed batch experiments were performed to test the effects of electron donor and electron acceptor availability on the production of chloroform (CF) during carbon tetrachloride (CT) destruction by a denitrifying bacterial consortium. In one series of tests, acetate (electron donor) was present in excess while nitrate and nitrite (electron acceptor) were limiting. In the other series of tests, acetate was the limiting nutrient, and nitrate and nitrite were in excess. Under nitrate limiting conditions, 50% (+/-17%) of the CT transformed by the microorganisms was converted to CF. However, under acetate limiting conditions, only 4% (+/-4%) of the CT that was degraded appeared as CF. Previous research had suggested that denitrifying bacteria can degrade CT via two competing pathways. One of these pathways produces CF as the predominant end product. The second pathway produces CO(2) as the primary end product. The results shown here suggest that the first pathway is dominant when nitrate and nitrite are depleted while the second pathway, which produces little CF, dominates when nitrate or nitrite are available.     

Improving protein extraction yield in reversed micellar systems through surface charge engineering

The mechanism of extraction of rat cytochrome b(5) from water into a sodium dioctylsulfosuccinate (AOT) micellar organic phase was studied using protein engineering of surface charged residues. The extraction behavior of native cytochrome b(5) and modified proteins with substitutions of the type glutamic acid --> lysine at positions 44 (E44K), 56 (E56K), and 92 (E92K), was studied as a function of pH. The results indicate that an important mechanism of extraction is an electrostatic interaction of this protein with the negatively charged surfactant. We demonstrate that it is possible to improve extraction by engineering the protein surface charge, increasing the driving force responsible for the protein transfer to the micellar phase. (c) 1994 John Wiley & Sons, Inc.     

Description of Ulvella elegans sp. nov. and U. islandica sp. nov. (Ulvellaceae,Ulvophyceae) from Iceland – a study based on morphology of species in culture and tufA gene sequences

Two new Ulvella species, U. elegans R. Nielsen & K. Gunnarsson and U. islandica R. Nielsen & K. Gunnarsson are described. These microfilamentous marine green algae were found in the sublittoral zone in northern Iceland, epiphytic on Euthora cristata and associated with a calcareous polychaete tube, respectively. Unialgal cultures were established from field-collected material for morphological observations. In culture, Ulvella elegans was characterized by rosettes of monostromatic pseudoparenchyma consisting of radiating filaments with a margin of mutually free filaments. Each cell had one pyrenoid. Hairs were not observed. Ulvella islandica had a heterotrichous morphology, consisting of dense tufts of upright broad branches and much narrower, rhizoid-like branches. Acrochaete-type hairs occurred; these are hyaline non-septate merocytic extensions from a more or less bulbous base, which may be separated from the vegetative cell below. Most cells had one pyrenoid except for a few broad cells which had two or three. In a phylogenetic reconstruction based on the chloroplast-encoded tufA gene, the sequences for the two species were clearly distinct from any other Ulvella sequence available for this gene. Ulvella islandica was placed in a clade together with U. lens, U. wittrockii, U. reticulata and U. pseudorepens. Ulvella elegans occupied a branch deep in the phylogeny but the position was poorly supported.     

The etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus HERV-Fc1

We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.     

Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells

The production of reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of hepatic diseases involving oxidative stress such as alcoholic liver disease (ALD). One consequence of ALD is increased insulin resistance in hepatocytes. To understand the effects of 4-HNE on insulin signaling in liver cells, we employed a model using hepatocellular carcinoma cell line HepG2. Previously, we have demonstrated an increase in the level of Akt phosphorylation is mediated by 4-HNE inhibition of PTEN, a direct regulator of Akt. In this work, we evaluated the effects of 4-HNE on insulin-dependent stimulation of the Akt2 pathway. We demonstrate that 4-HNE selectively leads to phosphorylation of Akt2. Although Akt2 is phosphorylated following 4-HNE treatment, the level of downstream phosphorylation of Akt substrates such as GSK3β and MDM2 is significantly decreased. Pretreatment with 4-HNE prevented insulin-dependent Akt signaling and decreased intracellular Akt activity by 87%. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of Akt2, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt2 (rAkt2) with 20 or 40 μM 4-HNE inhibited rAkt2 activity by 30 or 85%, respectively. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF) identified Michael addition adducts of 4-HNE with His196, His267, and Cys311 of rAkt2. Computation-based molecular modeling analysis of 4-HNE adducted to His196 and Cys311 of Akt2 suggests inhibition of GSK3β peptide binding by 4-HNE in the Akt2 substrate binding pocket. The inhibition of Akt by 4-HNE provides a novel mechanism for increased insulin resistance in ALD. These data provide a potential mechanism of dysregulation of Akt2 during events associated with sustained hepatocellular oxidative stress.