Identification of cytoplasmic and membrane-associated complexes in human embryonic stem cells using blue native PAGE

Human embryonic stem cells (hESCs) have great potential for use in developmental biology studies, functional genomics applications, drug screening, and regenerative medicine. A detailed understanding of the molecular mechanisms that are responsible for maintaining the undifferentiated and pluripotent nature of hESCs is essential for their effective therapeutic application. It has become evident that many complex cellular processes are carried out by assemblies of protein molecules (protein complexes). Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to separate protein complexes from whole cell lysates. Using BN-PAGE, we resolved cytoplasmic and membrane-associated complexes from hESCs and characterised their composition, stoichiometry, and dynamics by denaturing SDS-PAGE. The reliability of the fractionation was examined by western blot analysis of membrane and cytosolic markers. MALDI TOF/TOF mass spectrometry identified 119 cytosolic and 69 membrane proteins from the BN-PAGE proteome maps. Potential protein complexes were validated by computational prediction of possible protein-protein interactions using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Based on BN-PAGE gels and validation by databases, 82 heteromultimeric and 47 homomultimeric protein complexes have been found in hESCs. Resolving some of the protein complexes provided insight into the function of previously uncharacterised complexes in hESCs.     

Age as a Criterion for Setting Priorities in Health Care? A Survey of the German Public View

Although the German health care system has budget constraints similar to many other countries worldwide, a discussion on prioritization has not gained the attention of the public yet. To probe the acceptance of priority setting in medicine, a quantitative survey representative for the German public (n = 2031) was conducted. Here we focus on the results for age, a highly disputed criterion for prioritizing medical services. This criterion was investigated using different types of questionnaire items, from abstract age-related questions to health care scenarios, and discrete choice settings, all performed within the same sample. Several explanatory variables were included to account for differences in preference; in particular, interviewee''s own age but also his or her sex, socioeconomic status, and health status. There is little evidence that the German public accepts age as a criterion to prioritize health care services.     

Über die einwirkung von Röntgenstrahlen auf Amphibienlarven

Ohne Zusammenfassung Mit 22 Textabbildungen.     

Growth enhancement induced by prolonged L-dopa administration in rats

To follow growth of rats, in which growth hormone secretion has been chronically stimulated, L-Dopa (5 mg/kg) was injected subcutaneously twice daily for 70 days to growing rats. A control group, matched for sex and sibship, pair fed with the treatment group was given saline injections. At 10-day intervals, the rats were weighed and measured. At 90 days of age the rats were ether anesthesized, bled for growth hormone determination, and internal viscera weighed. Weight gain and length in the L-Dopa-treated group was found to be significantly greater. A mean weight gain of 6% and 12% in the male and female rats, respectively, and a mean length gain of 5% in both male and female rats was observed at 90 days of age. The thymus, thyroid, adrenals, uterus, and gonads all tended to be heavier in the L-Dopa-treated group. Significantly heavier kidneys were found in the L-Dopa group. Serum growth hormone was found to be 8.44 +/- 1.4(SE) ng/ml in the L-Dopa group and 4.6 +/- 0.9 ng/ml in the control group. It is concluded that the continuous administration of L-Dopa produces an increase of circulating serum growth hormone levels, and this in turn enhances growth.     

Imaging of alkaline phosphatase activity in bone tissue

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with (19)Flourine magnetic resonance spectroscopic imaging ((19)FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19)Fluorine magnetic resonance spectroscopy ((19)FMRS) and (19)FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19)FMRS and (19)FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19)FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19)FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19)FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19)FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.     

Gene expression programs during Brassica oleracea seed maturation, osmopriming, and germination are indicators of progression of the germination process and the stress tolerance level

During seed maturation and germination, major changes in physiological status, gene expression, and metabolic events take place. Using chlorophyll sorting, osmopriming, and different drying regimes, Brassica oleracea seed lots of different maturity, stress tolerance, and germination behavior were created. Through careful physiological analysis of these seed lots combined with gene expression analysis using a dedicated cDNA microarray, gene expression could be correlated to physiological processes that occurred within the seeds. In addition, gene expression was studied during early stages of seed germination, prior to radicle emergence, since very little detailed information of gene expression during this process is available. During seed maturation expression of many known seed maturation genes, such as late-embryogenesis abundant or storage-compound genes, was high. Notably, a small but distinct subgroup of the maturation genes was found to correlate to seed stress tolerance in osmoprimed and dried seeds. Expression of these genes rapidly declined during priming and/or germination in water. The majority of the genes on the microarray were up-regulated during osmopriming and during germination on water, confirming the hypothesis that during osmopriming, germination-related processes are initiated. Finally, a large group of genes was up-regulated during germination on water, but not during osmopriming. These represent genes that are specific to germination in water. Germination-related gene expression was found to be partially reversible by physiological treatments such as slow drying of osmoprimed seeds. This correlated to the ability of seeds to withstand stress.     

A concise method for the preparation of deuterium-labeled cortisone: synthesis of [6,7-2H]cortisone

A method is described for the synthesis of isotopically labeled cortisone from commercially available cortisone acetate through a Delta(4,6)-dieneone. Direct deuteration of the dienone acetate with various catalysts in different solvent systems failed to give an isolable product. Initial hydrolysis of the side-chain ester of the Delta(4,6)-dieneone and subsequent derivatization gave the key intermediate, 17alpha,20;20,21-bismethylenedioxy-pregna-4,6-diene-3,11-dione, which could be satisfactorily deuterated to the desired product. The availability of [6,7-(2)H]cortisone will provide a tool for the future study of the metabolism of cortisone in human tissues.