Acute hibernation decreases myocardial pyruvate carboxylation and citrate release

In the well-perfused heart, pyruvate carboxylation accounts for 3-6% of the citric acid cycle (CAC) flux, and CAC carbon is lost via citrate release. We investigated the effects of an acute reduction in coronary flow on these processes and on the tissue content of CAC intermediates. Measurements were made in an open-chest anesthetized swine model. Left anterior descending coronary artery blood flow was controlled by a extracorporeal perfusion circuit, and flow was decreased by 40% for 80 min to induce myocardial hibernation (n = 8). An intracoronary infusion of [U-(13)C(3)]lactate and [U-(13)C(3)]pyruvate was given to measure the entry of pyruvate into the CAC through pyruvate carboxylation from the (13)C-labeled isotopomers of CAC intermediates. Compared with normal coronary flow, myocardial hibernation resulted in parallel decreases of 65% and 79% in pyruvate carboxylation and net citrate release by the myocardium, respectively, and maintenance of the CAC intermediate content. Elevation of the arterial pyruvate concentration by 1 mM had no effect. Thus a 40% decrease in coronary blood flow resulted in a concomitant decrease in pyruvate carboxylation and citrate release as well as maintenance of the CAC intermediates.     

The sheep antibody response to repeated infection with Lucilia cuprina.

, , and 1992. The sheep antibody response to repeated infection with Lucilia cuprina. International Journal for Parasitology 22: 1169–1174. The specific serum antibody responses of sheep exposed to 10 consecutive infections of L. cuprina have been analysed by enzyme-linked immuno-sorbent assay and immunoblotting using monoclonal antibodies specific for sheep immunoglobulin isotypes. Recognition of a number of larval excretory-secretory products by IgM antibodies appeared to be non-specific. IgG₁ was the major antibody class stimulated by the infection protocol and marked increases in antibody to specific excretory-secretory antigens were observed. Three molecules of 35, 30 and 25 kDa were particularly recognized although the extent of recognition of these molecules varied considerably between individual sheep serum. A pooled serum composed of sera collected after five to seven infections significantly inhibited larval growth in in vitro cultures when compared to a sera pool consisting of sera collected both prior to infection and after infections 1 and 2. The degree of inhibition was greater when serum with high specific antibody titre was used.     

Complexation with heparin prevents adhesion between fibrin-coated surfaces.

Heparin, in Langmuirian fashion, binds stoichiometrically with high affinity, Kd approximately 100 nM, to both fibrinogen and fibrin adsorbed as monomolecular films to lecithin-coated, microscopic, polystyrene-divinylbenzene beads. Complex formation inhibits aggregation of fibrin-coated beads, and it also results in dissociation of preformed aggregates of fibrin-coated beads. These phenomena are not caused by desorption of fibrin(ogen), indirect inhibition of thrombin activity, or mere electrostatic repulsion of charged particles. Instead, these data are consistent with the proposal that the complexed heparin interferes directly with dimer formation between fibrin molecules adsorbed to colliding beads. We describe these phenomena and their application to the development of sensitive analytical methods for quantitating heparin. Based on these observations, we also propose a role for endogenous heparin in the physiologic regulation of fibrin-mediated adhesion of surfaces.     

Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites.

The integration host factor of Escherichia coli (IHF) is a small, histone-like protein which participates in the integration of bacteriophage lambda into the E. coli chromosome and in a number of regulatory processes. Our recent footprinting analysis has shown that IHF binds specifically to the ends of the transposable element IS1, as well as to several sites within a short segment of the plasmid pBR322. We have extended our studies of the binding of the IHF molecule to these sites in vitro using a gel retardation assay. We report here that IHF bends the DNA upon binding, as judged from the strong cyclic dependence of the protein-induced mobility shift on the position of the binding site. Using cloned, synthetic ends of IS1 as substrates, we have found that some mutations within the conserved bases of the IHF consensus binding sequence abolish binding, and that alterations of the flanking sequences can greatly reduce IHF binding. The presence of multiple IHF sites on a single DNA fragment increases binding very little, indicating that IHF does not bind cooperatively in this complex. We discuss the possibility that DNA bending is related to the role IHF plays in forming and stabilizing nucleoprotein complexes, and suggest that bending at the IHF sites may be important to its diverse effects in the cell.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.