Mechanism of taxol-induced apoptosis in human SKOV3 ovarian carcinoma cells

Taxol is extensively used clinically for chemotherapy of patients with ovarian, breast, and lung cancer. Although taxol induces apoptosis of cancer cells, its exact mechanism of action is not yet known. To determine the mechanism of action of taxol in ovarian cancer, we tested the effects of the drug, on the human ovarian carcinoma cell line, SKOV3. We observed that taxol-induced apoptosis of these cells by phosphatidylserine (PS) externalization and DNA fragmentation. While treatment of cells with taxol resulted in bcl-2 phosphorylation and mitochondrial depolarization, cytochrome c was not released and pro-caspase-3 was not activated. Treatment of SKOV3 cells with taxol, however, resulted in the translocation of AIF from the mitochondria to the nucleus via the cytosol. Taken together, these findings suggest that in SKOV3 cells, taxol induces caspase-independent AIF-dependent apoptosis.     

Oligonucleotides in human urine do not contain 8-oxo-7,8-dihydrodeoxyguanosine

The promutagenic DNA modification 8-oxo-7,8-dihydrodeoxyguanosine is the most frequently used marker for oxidative stress to DNA. The unmodified base and nucleoside and the 8-hydroxylated guanine base and nucleoside are found in urine, the latter used as a global measure of oxidative stress to DNA. Nucleotide excision repair (NER) excises a 27- to 29-mer oligonucleotide with oxidative lesions, and if found in urine, it could be used as a measure of DNA repair in vivo. Enzymatic hydrolysis of human urines followed by HPLC-tandem mass spectrometry was not able to reveal oligonucleotides and/or mononucleotides with the 8-oxo-7,8-dihydrodeoxyguanosine modification. The recovery of a synthetic oligonucleotide with the modification was complete (95% confidence limits: 98-124%). These experiments show that oligonucleotides are excreted into urine, but that 8-oxo-7,8-dihydrodeoxyguanosine is found only as the mononucleoside and is not present in any significant amounts in oligonucleotides. We conclude that oligonucleotides are excreted into urine, and they do not contain oxidized lesions. Either NER products are degraded after excision or NER functions differently in vivo in humans compared with cellular systems.     

Applying proteomics to signaling networks

The information from genome sequencing provides a new framework for a systems-wide understanding of protein networks and cellular function. Whereas microarray technologies provide information about global gene expression within cells, complementary proteomic strategies monitor expression of proteins and their posttranslational modifications. Improved technologies that have emerged for comprehensive and high-throughput protein analysis yield novel insights into cell regulation.     

Rapid growth and elongation of bovine blastocysts in vitro in a three-dimensional gel system

The aim of this study was to establish an in vitro system that supports development and differentiation of bovine blastocysts. Agar gel tunnels were covered with modified synthetic oviduct fluid medium supplemented with 10% fetal calf serum and 3g/l D-glucose. Of the total 67 blastocysts loaded individually into the tunnels, 46 continued expansion to 1mm and reached the walls of the gel on Day 10. On Day 12, 35 blastocysts elongated to minimum 1.6 mm while filling completely the space between the walls of the tunnel, and 16 still continued growth and reached an average of 4.3 mm length on Day 14. The largest blastocyst on Day 16 was 12 mm long. On Day 12, in 31 of the 35 elongated blastocysts a second cell layer occurred beneath the trophoblast and formed a complete cover in surviving Day 14 embryos. In most proliferating embryos the inner cell mass was prominent, however, the detection of signs of embryonic disc formation will require further studies. The established system was suitable to induce in vitro elongation, rapid growth and further differentiation, and may have considerable theoretical and practical value for studies of development and differentiation of bovine embryos.     

Detection and characterization of a dehalogenating microorganism by terminal restriction fragment length polymorphism fingerprinting of 16S rRNA in a sulfidogenic, 2-bromophenol-utilizing enrichment

Terminal restriction fragment length polymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment length polymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (a Desulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a second Desulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment length polymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.     

Effects of climate on incidence of Campylobacter spp. in humans and prevalence in broiler flocks in Denmark

Campylobacter infections are increasing and pose a serious public health problem in Denmark. Infections in humans and broiler flocks show similar seasonality, suggesting that climate may play a role in infection. We examined the effects of temperature, precipitation, relative humidity, and hours of sunlight on Campylobacter incidence in humans and broiler flocks by using lag dependence functions, locally fitted linear models, and cross validation methods. For humans, the best model included average temperature and sunlight 4 weeks prior to infection; the maximum temperature lagged at 4 weeks was the best single predictor. For broilers, the average and maximum temperatures 3 weeks prior to slaughter gave the best estimate; the average temperature lagged at 3 weeks was the best single predictor. The combined effects of temperature and sunlight or the combined effects of temperature and relative humidity predicted the incidence in humans equally well. For broiler flock incidence these factors explained considerably less. Future research should focus on elements within the broiler environment that may be affected by climate, as well as the interaction of microclimatic factors on and around broiler farms. There is a need to quantify the contribution of broilers as a source of campylobacteriosis in humans and to further examine the effect of temperature on human incidence after this contribution is accounted for. Investigations should be conducted into food consumption and preparation practices and poultry sales that may vary by season.     

Enhanced immunodetection of cell-free synthesized erythropoietin under denaturing conditions

A monoclonal antibody produced by hydridoma cell line, ATCC HB8209, was used to detect and purify erythropoietin synthesized in a cell-free system. The antibody was raised against the N-terminal 20 residues of erythropoietin. It retained anti-erythropoietin activity in 6 M urea in which most of the cell-free synthesized erythropoietin became soluble and gave an enhanced activity of the antibody.     

Quantitative analysis of bacterial communities from Mediterranean sapropels based on cultivation-dependent methods

Microbial communities of ancient Mediterranean sapropels, buried sediment layers of high organic matter, were analyzed by most probable number (MPN) approaches. Mineral media containing different carbon sources in sub-millimolar concentrations were used. MPN numbers were elevated in sapropels and at the sediment surface, which mirrored total cell count distributions. Highest MPN counts were obtained with a mixture of different monomeric and polymeric substrates, with amino acids or with long-chain fatty acids as sole carbon sources. These values reached up to 2 x 10(7) cm(-3), representing 3.3% of the total cell count. A total of 98 pure cultures were isolated from the highest positive dilutions of the MPN series, representing the most abundant microorganisms culturable by the methods used. The strains were identified by molecular biological methods and could be grouped into 19 different phylotypes. They belonged to the alpha-, beta-, gamma-, and delta-Proteobacteria, to the Actinobacteria and the Firmicutes. However, about half of the number of isolates was closely related to the genera Photobacterium and Agrobacterium. Regarding the high cultivation success, these organisms can be assumed to be typical sapropel bacteria, representing a substantial part of the culturable indigenous microbial community.