Genomic Stability of Lyophilized Sheep Somatic Cells before and after Nuclear Transfer

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.     

Bacterial lipolytic activity in a hypertrophic dead arm of the river Danube in Vienna

The kinetic parameters of lipase, bacterial secondaryproduction (BSP) and bacterial numbers (BN) were determined fortnightlyduringthe development of the summer phytoplankton bloom at twostationsof Alte Donau, a hypertrophic stagnant dead arm of the riverDanubein Vienna. Until the middle of August we observed a gradualincrease in lipase activity as well as BN and BSP rates tothe maximum of 19.9 nmol l^–1 h^–1,4.5×10⁹cells l^–1 and 8.1 g C l^–1 h^–1,respectively. Atthe end of August and during September we found a markeddecreasein all bacterial parameters, coinciding with a progressingincreaseof chlorophyll a concentrations at both sampling sites. Themaximalvalues of lipase V^max were determined in the bottom waterlayer (avg. 13.7±6.5 nmol l^–1 h^–1) probablyowingto the predominating importance of polymeric matter in thesubstrate pool for microheterotrophs in this water zone.Differential filtration experiments showed that 20.1% to56.3% ofthe total lipase activity and 4.2% to 9.0% of the totalbacterialnumbers in Alte Donau water samples occurred in 0.2-mfiltrate. Further experiments indicated that the highcontributionto lipase activity in the 0.2-m filtrate was rather dueto thepresence of 0.2 m filterable bacteria than to solubleenzymemolecules. Moreover, we observed higher bacterial lipaseactivityin 0.2 m filtrate than in unfiltered samples. Thepossibleinfluence of limiting factors on the metabolism of insitubacteria is discussed.     

Regional specialization of the sperm membrane in the tick Amblyomma hebraeum koch (Acari: Ixodidae)

Summary The spermatozoon of Amblyomma hebraeum is about 200 m long and comprises: (1) a thick, club-shaped anterior part, about 20 m long bearing at its apex a tactile hemisphere, and (2) an elongated tail-like part, about 180 m long. The surface of the tactile hemisphere is covered by numerous bulbous expansions, attached to it by short stalks. The base of the hemisphere is surrounded by a fringe of thin motile processes; the remaining surface of the spermatozoon is covered with long cellular processes which run more or less parallel to one another.The membrane-associated particles found on the membrane beneath the cellular processes are regularly arranged as groups of parallel strands. The external surface of the so-called peripheral granules, as revealed by freeze-etching, is smooth with a very small number of particles. Internally the particles exhibit a regular hexagonal pattern which has not been observed, so far, on any other membrane of these sperm cells.The regional specialization of the spermatozoon surface membrane in relation to sperm motility is discussed. The results obtained indicate that processes of three types: (1) bulbous expansions, (2) motile processes, and (3) cellular processes are regional specializations, all engaged in aspects of sperm motility.The technical assistance of Mr. R. Haemmerle of Balzers Research Laboratories, Mrs. F. Seif and Miss C. Pugin, is gratefully acknowledged     

Phenotypic and functional analysis of murine CD3+,CD4-,CD8- TCR-gamma delta-expressing peripheral T cells

Murine CD3+,CD4-,CD8- peripheral T cells, which express various forms of the TCR-gamma delta on their cell surface, have been characterized in terms of their cell-surface phenotype, proliferative and lytic potential, and lymphokine-producing capabilities. Three-color flow cytofluorometric analysis demonstrated that freshly isolated CD3+,CD4-, CD8- TCR-gamma delta lymph node cells were predominantly Thy-1+,CD5dull,IL-2R-,HSA-,B220-, and approximately 70% Ly-6C+ and 70% Pgp-1+. After CD3+,CD4-,CD8-splenocytes were expanded for 7 days in vitro with anti-CD3-epsilon mAb (145-2C11) and IL-2, the majority of the TCR-gamma delta cells expressed B220 and IL-2R, and 10 to 20% were CD8+. In comparison to CD8+ TCR-alpha beta T cells, the population of CD8+ TCR-gamma delta-bearing T cells exhibited reduced levels of CD8, and about 70% of the CD8+ TCR-gamma delta cells did not express Lyt-3 on the cell surface. Functional studies demonstrated that splenic TCR-gamma delta cells proliferated when stimulated with mAb directed against CD3-epsilon, Thy-1, and Ly-6C, but not when incubated with an anti-TCR V beta 8 mAb, consistent with the lack of TCR-alpha beta expression. In addition, activated CD3+,CD4-,CD8- peripheral murine TCR-gamma delta cells were capable of lysing syngeneic FcR-bearing targets in the presence of anti-CD3-epsilon mAb and the NK-sensitive cell line, YAC-1, in the absence of anti-CD3-epsilon mAb. Finally, activated CD3+, CD4-,CD8-,TCR-gamma delta+ splenocytes were also capable of producing IL-2, IL-3, IFN-gamma, and TNF when stimulated in vitro with anti-CD3-epsilon mAb.     

Damage to the DNA bases in mammalian chromatin by hydrogen peroxide in the presence of ferric and cupric ions.

Modification of DNA bases in mammalian chromatin upon treatment with hydrogen peroxide in the presence of ferric and cupric ions was studied. Ten DNA base products in mammalian chromatin were identified and quantitated by the use of gas chromatography-mass spectrometry with selected-ion monitoring after hydrolysis of chromatin and trimethylsilylation of hydrolysates. This technique permitted the analysis of modified DNA bases in chromatin without the necessity of isolation of DNA from chromatin first. Modified bases identified were typical hydroxyl radical-induced products of DNA, indicating the involvement of hydroxyl radical in their formation. This was also confirmed by inhibition of product formation by typical scavengers of hydroxyl radical. The inhibition of product formation was much more prominent in the presence of chelated ions than unchelated ions, indicating a possible site-specific formation of hydroxyl radical when metal ions are bound to chromatin. Hydrogen peroxide in the presence of cupric ions caused more DNA damage than in the presence of ferric ions. Chelation of cupric ions caused a marked inhibition in product formation. By contrast, DNA was damaged more extensively in the presence of chelated ferric ions than in the presence of unchelated ferric ions. The presence of ascorbic acid generally increased the yields of the products, indicating increased production of hydroxyl radical by reduction of metal ions by ascorbic acid. Superoxide dismutase afforded partial inhibition of product formation only in the case of chelated iron ions. The yields of the modified bases in chromatin were lower than those observed with calf thymus DNA under the same conditions.