Is apoptosis in bovine in vitro produced embryos related to early developmental kinetics and in vivo bull fertility?

Although several studies have indicated a paternal effect on bovine embryo development, no conclusive data exist on the effect of in vivo bull fertility on apoptosis. Therefore, it was the main objective of this study to compare the apoptotic cell ratio (ACR) in embryos originating from bulls with different in vivo fertility. However, since it is has been demonstrated before that bulls with different in vivo fertility differ in timing of first cleavage, it was necessary to investigate first the effect of timing of development on apoptosis in vitro in order to get an unbiased insight in the contribution of in vivo bull fertility on apoptosis in bovine blastocysts. In the first experiment, bovine embryos (n = 939) were allocated to different groups according to cleavage rate at 30, 36 and 48 hpi and blastocysts were selected at 7 and 8 dpi. The blastocyst rate at 7 dpi was significantly lower in embryos which had first cleaved at 48 hpi than in embryos from the 30 and 36 hpi group (P < 0.05). The ACR after TUNEL in day 7 blastocyst was significantly lower in the 30 hpi group in comparison with the 36 and 48 hpi group (P < 0.05) and lower in day 7 blastocysts than in day 8 blastocysts. In the second experiment, sperm of eight bulls with different non return rates was used for in vitro bovine embryo production (n = 3820 oocytes). Cleavage rates (30, 36 and 48 hpi) and blastocyst rate (7 dpi) were determined. Only very low negative correlations could be found between in vivo and in vitro bull fertility and ACR did not differ between groups derived from sires with either low or normal fertility (P > 0.05). Further research in serum free conditions is needed to confirm that the lower ACR in early cleaved embryos could be mediated by the cooperative interaction of embryos of good quality cultured in group. In vivo bull fertility could hardly be correlated with in vitro blastocyst yield and could not be correlated with appearance of apoptosis.     

Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.     

A vital staining method for measuring the efficiency of transfection of eucaryotic cells

The xylE gene encodes catechol 2,3-dioxygenase, which catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. The expression of this gene in eucaryotic cells can be detected simply by addition of catechol to the growth medium of the cells: cells that have a sufficient level of expression of the xylE gene stain yellow because of the accumulation of 2-hydroxymuconic semialdehyde. The number of stained cells is thus dependent upon the transfection efficiency as well as the level of expression of the xylE gene and is a measure of the combined transfection/expression efficiency in a particular cell type. Since the staining procedure does not affect the viability of the culture, the cells can be harvested afterward and analyzed for the expression of other, cotransfected, genes. This system for measuring transfection efficiency is especially useful when only small amounts of tissue are available.     

mRNA expression of Bcl-2, Bax, caspase-3 and -7 cannot be used as a marker for apoptosis in bovine blastocysts

At present the most widely used technique for apoptosis detection in embryos remains the in situ visualization of DNA fragmentation by terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) assay although this technique may be prone to artefacts. The aim of the present study was to investigate if the mRNA expression of a set of genes involved in apoptosis (Bax, Bcl-2, caspase-3 and -7) at an earlier point in the apoptotic cascade could be a good marker for apoptosis in in vitro produced bovine embryos. After normalization to the geometric mean of three reference genes, GAPD, YWHAZ and SDHA, mRNA expression levels of Bax, Bcl-2, caspase-3 and -7 were compared in embryos treated with an apoptosis inducer, staurosporine and in non-treated embryos. None of the genes were differently expressed in treated in comparison with non-treated embryos. In conclusion, mRNA expression of Bax, Bcl-2, caspase-3 and-7 cannot be used as a reliable apoptosis detection method. Immunofluorescent staining of caspase-3 and -7 is a better choice where as for Bcl-2 no reliable and practicable alternative is available at the moment.     

Human herpesvirus 6 infection arrests cord blood mononuclear cells in G(2) phase of the cell cycle

We here report that after infection with human herpesvirus 6A, human cord blood mononuclear cells accumulate in G₂/M phase of the cell cycle. Experiments with foscarnet or ultraviolet (UV)-irradiated virus stocks pointed at an (immediate-)early, newly formed viral protein to be responsible for the arrest. At the molecular level, p53, cyclin B₁, cyclin A and tyrosine¹⁵-phosphorylated cdk1 accumulated after HHV-6A infection, indicating an arrest in G₂. However, no change was observed in the levels of downstream effectors of p53 in establishing a G₂ arrest, i.e. p21 and 14-3-3σ. We thus conclude that the HHV-6A-induced G₂ arrest occurs independently of p53 accumulation.     

Polyhydroxylated steroids from the soft coral Sinularia dissecta

A repeated silica gel column chromatography followed by HPLC purification on the methanol extract of marine soft coral Sinularia dissecta, resulted in the isolation of fifteen polyhydroxylated steroids (1-15), involving six new C-18 functionalized steroids, 3beta-acetoxy-1alpha,11alpha-dihydroxygorgost-5-en-18-oic acid (1), gorgost-5-en-1alpha,3beta,11alpha,18-tetrol (2), 18-acetoxy-1alpha,3beta,11alpha-trihydroxygorgost-5-ene (3), 24(S)-3beta-acetoxy-1alpha, 11alpha-dihydroxyergost-5-en-18-oic acid (4), 24(S)-ergost-5-en-1alpha,3beta,11alpha,18-tetrol (5), and dissectolide (6). The structures of the new compounds were determined on the basis of extensive spectroscopic data (IR, MS, (1)H and (13)C NMR, HMQC, HMBC, and NOESY) analysis. Compound 6 was found as an unusual sterol bearing a lactone functionality.     

Evolutionary history of the closely related group 2 coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus OC43

The close genetic and antigenic relatedness among the group 2 coronaviruses human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and porcine hemagglutinating encephalomyelitis virus (PHEV) suggests that these three viruses with different host specificities diverged fairly recently. In this study, we determined the complete genomic sequence of PHEV (strain PHEV-VW572), revealing the presence of a truncated group 2-specific ns2 gene in PHEV in comparison to other group 2 coronaviruses. Using a relaxed molecular clock approach, we reconstructed the evolutionary relationships between PHEV, BCoV, and HCoV-OC43 in real-time units, which indicated relatively recent common ancestors for these species-specific coronaviruses.     

Receptor and antibody interactions of human interleukin-3 characterized by mutational analysis.

Human interleukin-3 (hIL-3) is a regulator of proliferation and differentiation of multipotent hemopoietic progenitor cells. Mutants of hIL-3 have been constructed by oligonucleotide-directed mutagenesis and expressed in Escherichia coli and Bacillus licheniformis. Purified muteins were assayed for induction of DNA synthesis in IL-3-dependent human cells and for binding to the IL-3 receptor. Residues at the NH2 and COOH termini together comprising one-quarter of the molecule could be removed without loss of biological function. Deletions of 6-15 residues within the central part of the molecule caused a large reduction (up to 5 logs) but no complete loss of activity. Substitution of evolutionary conserved residues resulted in a strong decrease of biological activity and demonstrated that the S-S bridge is an essential structural element in hIL-3. Interestingly, four muteins displayed a significantly higher potency of binding to the IL-3 receptor than in stimulating DNA synthesis. These results demonstrate that receptor binding may be (partly) disconnected from activation of DNA synthesis. Analysis of hIL-3 muteins demonstrated that the majority of monoclonal antibodies are directed against a small portion of the IL-3 molecule. The neutralizing potential of individual monoclonal antibodies could be increased by a combination of antibodies directed against nonoverlapping epitopes.