Proteomic Interrogation of Human Chromatin

Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the “Chromatome”) is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes

Intermittent Ischemia: An Alternative to Cardioplegic Arrest during Myocardial Revascularization Surgery

Cardioplegic myocardial protection has become the standard for myocardial revascularization surgery (MRS). In contrast, our group performed 500 consecutive MRS' with intermittent aortic cross-clamping for distal anastomoses, left ventricular venting, and systemic hypothermia. Average patient age was 62 years (range: 30-89 years). 194 patients (38.8%) had urgent or emergent MRS. 251 patients (50.2%) had unstable angina and 123 others (24.6%) had preinfarction angina (rest pain in the hospital); 27 (5.4%) had evolving myocardial infarctions (MI). 174 patients (34.8%) had ejection fractions (EF) <0.50 including 75 patients (15.0%) with EFs <0.40; 16 patients (3.2%) had left ventricular aneurysms. Average number of grafts was 3.3 per patient and average ischemic time was 7.6 minutes per graft. There were five hospital deaths (1.0%), none due to poor myocardial protection and low cardiac output. Only three survivors (0.6%) required an intra-aortic balloon pump (IABP) to wean from cardiopulmonary bypass (CPB): two had acute MIs preoperatively; the other had EF <0.30 and intractable atrial arrhythmias. Only two other patients (0.4%) received any inotropic infusions postoperatively. 18 patients (3.6%) had perioperative MIs. These results, particularly the virtual absence of postoperative inotropic support, in unselected patients of whom 80% had acute coronary syndromes, indicate that intermittent ischemia (II) can provide excellent myocardial protection for MRS. Brief periods of II alleviate concerns about cardioplegic protection via occluded coronary arteries or internal mammary artery grafts. II provides a simple and safe alternative to cardioplegic arrest for myocardial protection during MRS

Evaluation of Three Types of Membrane Oxygenators and Their Suitability for Use with Pulsatile Flow

(J. Extra-Corpor. Technol. 19[3] p. 297-304 Fall 1987, 26 ref.) Three types of single-pumphead arterial line microporous polypropylene membrane oxygenators were evaluated: 1) flat plate; 2) hollow fiber with blood path inside the fibers; and 3) hollow fiber with blood path outside the fibers. These oxygenator types were evaluated to determine what hemodynamic effects they would have on the pulse waveform in the extracorporeal arterial line and the radial artery monitoring line of the patient during pulsatile cardiopulmonary bypass. Gaseous microemboli (GME) generated by these oxygenators during pulsatile cardiopulmonary bypass were also measured. Hemodynamically, the flat plate type exhibited the greatest tendency to dampen the pulsatile waveform because it is the most compliant membrane of the three types evaluated. The hollow fiber with blood path inside the fibers type had the highest line pressures proximal to the membrane and low pressure distal to the membrane because it is the most resistant to flow (i.e., high resistance across the membrane). The hollow fiber with blood path outside the fibers type had the least effect on the transmission of the pulsatile waveform because it has very low resistance across the membrane and very little compliance. With regard to gaseous microemboli, the flat plate type and the hollow fiber with blood path outside the fibers type had negligible counts, during both continuous flow and pulsatile flow. The hollow fiber with blood path inside the fibers type had somewhat elevated gaseous microemboli counts, especially during initiation of pulsatile cardiopulmonary bypass; however, these diminished with time. We conclude that the hollow fiber with blood path outside the fibers type is the type oxygenator of choice when employing pulsatile cardiopulmonary bypass with an arterial line membrane oxygenator

The emerging role of GABAB receptors as regulators of network dynamics: fast actions from a ‘slow’ receptor?

Convention holds that ionotropic receptors mediate fast neurotransmission and that ‘slow’ G-protein coupled metabotropic receptors have a secondary, modulatory role in the control of neuronal networks. Here, we discuss recent evidence showing that activation of metabotropic GABA(B) receptors in cortical layer 1 can powerfully inhibit principal cell activity and that their activation can rapidly halt ongoing network activity. Inputs from both within and outwith the cortex converge upon layer 1 where they target various populations of interneurons, including neurogliaform cells. We argue that neurogliaform cells are the main effector of a powerful inhibitory circuit that, acting through GABA(B) receptors, can be differentially recruited by long-range connections to serve in roles as diverse as conscious perception and memory consolidation

A Link between Aurora Kinase and Clp1/Cdc14 Regulation Uncovered by the Identification of a Fission Yeast Borealin-Like Protein

The chromosomal passenger complex (CPC) regulates various events in cell division. This complex is composed of a catalytic subunit, Aurora B kinase, and three nonenzymatic subunits, INCENP, Survivin, and Borealin. Together, these four subunits interdependently regulate CPC function, and they are highly conserved among eukaryotes. However, a Borealin homologue has never been characterized in the fission yeast, Schizosaccharomyces pombe. Here, we isolate a previously uncharacterized S. pombe protein through association with the Cdc14 phosphatase homologue, Clp1/Flp1, and identify it as a Borealin-like member of the CPC. Nbl1 (novel Borealin-like 1) physically associates with known CPC components, affects the kinase activity and stability of the S. pombe Aurora B homologue, Ark1, colocalizes with known CPC subunits during mitosis, and shows sequence similarity to human Borealin. Further analysis of the Clp1–Nbl1 interaction indicates that Clp1 requires CPC activity for proper accumulation at the contractile ring (CR). Consistent with this, we describe negative genetic interactions between mutant alleles of CPC and CR components. Thus, this study characterizes a fission yeast Borealin homologue and reveals a previously unrecognized connection between the CPC and the process of cytokinesis in S. pombe