HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Novel Approach to Simulate Sleep Apnea Patients for Evaluating Positive Pressure Therapy Devices

Bench testing is a useful method to characterize the response of different automatic positive airway pressure (APAP) devices under well-controlled conditions. However, previous models did not consider the diversity of obstructive sleep apnea (OSA) patients’ characteristics and phenotypes. The objective of this proof-of-concept study was to design a new bench test for realistically simulating an OSA patient’s night, and to implement a one-night example of a typical female phenotype for comparing responses to several currently-available APAP devices. We developed a novel approach aimed at replicating a typical night of sleep which includes different disturbed breathing events, disease severities, sleep/wake phases, body postures and respiratory artefacts. The simulated female OSA patient example that we implemented included periods of wake, light sleep and deep sleep with positional changes and was connected to ten different APAP devices. Flow and pressure readings were recorded; each device was tested twice. The new approach for simulating female OSA patients effectively combined a wide variety of disturbed breathing patterns to mimic the response of a predefined patient type. There were marked differences in response between devices; only three were able to overcome flow limitation to normalize breathing, and only five devices were associated with a residual apnea-hypopnea index of <5/h. In conclusion, bench tests can be designed to simulate specific patient characteristics, and typical stages of sleep, body position, and wake. Each APAP device behaved differently when exposed to this controlled model of a female OSA patient, and should lead to further understanding of OSA treatment.     

Vererbungsstudien

Ohne Zusammenfassung     

Mitotic drug targets

Mitosis is the key event of the cell cycle during which the sister chromatids are segregated onto two daughter cells. It is well established that abrogation of the normal mitotic progression is a highly efficient concept for anti‐cancer treatment. In fact, various drugs that target microtubules and thus interfere with the function of the mitotic spindle are in clinical use for the treatment of various human malignancies for many years. However, since microtubule inhibitors not only target proliferating cells severe side effects limit their use. Therefore, the identification of novel mitotic drug targets other than microtubules have gained recently much attention. This review will summarize the latest developments on the identification and clinical evaluation of novel mitotic drug targets and will introduce novel concepts for chemotherapy that are based on recent progress in our understanding how mitotic progression is regulated and how anti‐mitotic drugs induce tumor cell death. J. Cell. Biochem. 111: 258–265, 2010. © 2010 Wiley‐Liss, Inc.     

Immunocytochemical detection of p21ras expression in fresh human leukaemic cells and cell lines

The expression of p21ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21ras could be detected in most of the cell lines, no significant p21ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state.     

Kinetic β-deuterium isotope effects suggest a covalent mechanism for the protein folding enzyme peptidylprolyl cis/trans-isomerase

The cis/trans interconversion of Glt-Ala-Ala-Pro-Phe-4-nitroanilide and Glt-Ala-Gly-Pro-Phe-4-nitroanilide was studied both enzymatically and nonenzymatically by measuring kinetic β-deuterium isotope effects. The hydrogen atom at the α-carbon atom of the Xaa residue within the Xaa-Pro moiety was substituted by deuterium. In the nonenzymatic case the transition state of rotation is reflected by k_H/k_D > 1. When catalysed by 17 kDa PPIase the same bond rotation is characterized by k_H/k_D < 1. This suggests a covalent mechanism of catalysis which involves an approximately tetravalent carbon of the prolyl imidic bond for the transition state of reaction.     

Cell kinetic disturbances induced by treatment of human diploid fibroblasts with 5-azacytidine indicate a major role for DNA methylation in the regulation of the chromosome cycle

Summary BrdU-Hoechst flow cytometry was used to investigate the effects of DNA hypomethylation, induced by treatment with 5-azacytidine (5AC), on cell proliferation. When human fibroblast-like cells derived from skin and amniotic fluid were exposed to 5AC during three successive cell cycles, their clone-forming ability was diminished after removal of the drug. Treated cells were rendered quiescent by culture with low serum in the absence of the drug. Upon serum stimulation, they showed a diminished fraction of proliferating cells, which exhibited a prolonged transit through the S and G2 phase of the cell cycle, and a permanent arrest within the G2 compartment. This pattern of disturbed cell proliferation may in part explain the changes in replication banding pattern reported in the literature. Cytogenetic analysis of 5AC-treated cells revealed numerous endomitoses and tetraploid metaphases indicating a disturbed chromosome cycle in association with these cell kinetic perturbations.