Histone Variants and Their Post-Translational Modifications in Primary Human Fat Cells

Epigenetic changes related to human disease cannot be fully addressed by studies of cells from cultures or from other mammals. We isolated human fat cells from subcutaneous abdominal fat tissue of female subjects and extracted histones from either purified nuclei or intact cells. Direct acid extraction of whole adipocytes was more efficient, yielding about 100 µg of protein with histone content of 60% –70% from 10 mL of fat cells. Differential proteolysis of the protein extracts by trypsin or ArgC-protease followed by nanoLC/MS/MS with alternating CID/ETD peptide sequencing identified 19 histone variants. Four variants were found at the protein level for the first time; particularly HIST2H4B was identified besides the only H4 isoform earlier known to be expressed in humans. Three of the found H2A potentially organize small nucleosomes in transcriptionally active chromatin, while two H2AFY variants inactivate X chromosome in female cells. HIST1H2BA and three of the identified H1 variants had earlier been described only as oocyte or testis specific histones. H2AFX and H2AFY revealed differential and variable N-terminal processing. Out of 78 histone modifications by acetylation/trimethylation, methylation, dimethylation, phosphorylation and ubiquitination, identified from six subjects, 68 were found for the first time. Only 23 of these modifications were detected in two or more subjects, while all the others were individual specific. The direct acid extraction of adipocytes allows for personal epigenetic analyses of human fat tissue, for profiling of histone modifications related to obesity, diabetes and metabolic syndrome, as well as for selection of individual medical treatments

Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures

In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets—pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches

Hydroxyethyl starch solution: Is it a new alternative way of treatment in bacterial translocation?

WOS: 000077220900017PubMed ID: 9870785In this experimental study, the effects of mannitol and hydroxyethyl starch solution (HES) on bacterial translocation were evaluated in a rat model of simple mechanical bowel obstruction. The mechanical bowel obstruction was created by ligation of the ileum 1 cm proximal to the ileocecal valve, Excluding the control group (n=7), the rats were given 2 mi of mannitol or 2 mi of 6% hydroxyethyl starch solution intraperitoneally during the procedure. Relaparotomy was made and segmental heal resection, total mesenteric lymph node excision and removal of tissue sample of liver were performed 24 h after the mechanical bowel obstruction, both for histopathological and microbiological examination. The rates of colony formation in the mesenteric lymph nodes after the mechanical bowel obstruction were 71% (5/7) in control animals, 43% (3/7) in animals given mannitol and only 14% (1/7) in animals given HES, In the liver samples observed for the bacterial translocation, the corresponding figures were 14% (1/7), 28% (2/7) and 0% (0/7) respectively, The difference between hydroxyethyl starch solution and control groups were statistically significant (P < 0.04), The bacterial population/g tissue obtained from intraluminal bacterial cultures of the ileum was also depressed in hydroxyethyl starch solution group when compared to the control (P < 0.03), As a result, we confirmed that hydroxyethyl starch solution, a plasma volume expander, depressed the bacterial translocation to the mesenteric lymph nodes and liver and also reduced intraluminal bacterial overgrowth in the simple mechanical bowel obstruction model of rats. We believe that hydroxyethyl starch solution may be an alternative therapeutic agent for the pre-operative fluid resuscitation in simple mechanical bowel obstruction