Quantum chemical calculations of the reorganization energy of blue-copper proteins

The inner-sphere reorganization energy for several copper complexes related to the active site in blue-copper protein has been calculated with the density functional B3LYP method. The best model of the blue-copper proteins, Cu(Im)2(SCH3)(S(CH3)2)(0/+), has a self-exchange inner-sphere reorganization energy of 62 kJ/mol, which is at least 120 kJ/mol lower than for Cu(H2O)4(+/2+). This lowering of the reorganization energy is caused by the soft ligands in the blue-copper site, especially the cysteine thiolate and the methionine thioether groups. Soft ligands both make the potential surfaces of the complexes flatter and give rise to oxidized structures that are quite close to a tetrahedron (rather than tetragonal). Approximately half of the reorganization energy originates from changes in the copper-ligand bond lengths and half of this contribution comes from the Cu-S(Cys) bond. A tetragonal site, which is present in the rhombic type 1 blue-copper proteins, has a slightly higher (16 kJ/mol) inner-sphere reorganization energy than a trigonal site, present in the axial type 1 copper proteins. A site with the methionine ligand replaced by an amide group, as in stellacyanin, has an even higher reorganization energy, about 90 kJ/mol.     

A comparison of thromboelastography with heparinase or protamine sulfate added in vitro during heparinized cardiopulmonary bypass

Thromboelastography (TEG) has been used after cardiopulmonary bypass (CPB) to diagnose excessive postoperative hemorrhage. Conventional TEG during CPB is not possible due to the sensitivity of the TEG to even small amounts of heparin, which produces a nondiagnostic tracing. The purpose of this study was to compare heparin neutralization using heparinase or protamine in TEG blood samples obtained during CPB. TEG testing was performed on 48 patients before, during and after CPB. Tissue plasminogen activator activity and antigen were measured on a subset of 32 patients. We found: 1) heparinase neutralized at least 10 IU/ml heparin while 1.6 ug/ml protamine neutralized up to 7 IU/ml heparin, 2) in samples with complete heparin neutralization by both methods, there was no significant difference in the R values, 3) while there was good correlation for other TEG parameters between heparinase and protamine treated samples, heparinase treatment produced shorter K values and higher angle, MA and A60, 4) while fibrinolysis was detected using both methods, heparinase treatment suppressed fibrinolysis in the TEG in both samples from patients and after in vitro addition of tissue plasminogen activator, 5) TEG was not a sensitive indicator of t-PA activity, detecting only 21% of samples with increased t-PA activity during bypass, and 5) heparinase was at least 100 times more expensive than protamine. We conclude that while both heparinase and protamine can be used to neutralize heparin in TEG samples obtained during CPB, protamine neutralization is more sensitive to fibrinolysis and less expensive, but the protamine dose must be carefully selected to match the heparin level used at individual institutions.     

Anti-laminin reactivity and glomerular immune deposition by in vitro recombinant antibodies

Growing evidence suggests that recombinatorial events prior to antigen contact can generate pathogenic autoantibodies in the nonautoimmune individual, thus providing potential disease mediators if conditions arise that permit bypass of tolerance and activation of autoreactive lymphocytes. To examine the disease potential of selected germline antibody genes, Ig were created de novo by in vitro recombination of Ig H and L chains. H chain loss variant (i.e., L-chain only) cell lines were transfected with a DNA construct encoding the variable region and regulatory sequences (LamH) of a nephrotropic murine lupus anti-laminin Ig, and the resultant Ig were examined for in vitro antigen reactivity and in vivo glomerular immune deposition. The results indicate that two light chains, LamL (Vk8, Jk5) and 238L (Vk4, Jk5), expressing unrelated germline V1 genes, combine with LamH to generate Ig that bind basement membrane laminin in vitro, diverge in their capacity to bind ssDNA, and produce two distinct patterns of glomerular immune deposits in vivo: dense mesangial matrix (LamH/LamL) and dramatic linear glomerular basement membrane (LamH/238L) deposits. The Ig genes used by both LamH and 238L are present in nonautoimmune mice as well as in lupus-prone strains. We conclude that certain unmutated Ig genes can contribute to multiple distinct disease associated specificities, including binding to intrinsic kidney antigens, and that mutation is not essential to generate these Ig. Collectively, these observations suggest that pathogenic autoantibodies can be generated in the normal preimmune repertoire by random recombinatorial and somatic events in the absence of mutation.     

Demonstration of protein-protein interaction specificity by NMR chemical shift mapping

Chemical shift mapping is becoming a popular method for studying protein-protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N)-HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non-specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.     

Promotion of healthy lifestyle: knowledge and attitudes of primary care physicians

Preretinal neovascularization and chronic retinal oedema are the two major sight-threatening complications that can occur during diabetic retinopathy. Ocular neovascularization is strongly associated with retinal ischaemia, and growth factors have been implicated in its pathogenesis. The ischaemic retina is assumed to secrete growth factors that stimulate residual vessels to proliferate. Interest has focused on basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta) and more recently vascular endothelial cell growth factor (VEGF). Histologic studies have demonstrated the presence of growth factor proteins and receptors and/or their mRNA, mainly VEGF, PDGF, and bFGF, in preretinal membranes of patients with proliferative diabetic retinopathy. Elevated intravitreal levels of IGF-1 and VEGF correlating with neovascular activity have been found in some patients. However, a direct causal relationship between ischaemia, growth factors and neovascularization has not been clearly demonstrated despite considerable research work. To date, the growth factor correlating most closely with neovascularization is VEGF. As many growth factors seem to be produced during the neovascular process, their specific inhibition probably will have limited effects. Laser photocoagulation of the retina has proved beneficial for regression of new vessels, probably through destruction of the ischaemic retina producing neovascular growth factors, and is currently the only treatment for proliferative diabetic retinopathy. Inhibition of IGF-1 by somatostatin analogs has produced unsatisfactory results. Other vascular inhibitors are currently being studied.     

Laser-assisted preparation of single cells from stained histological slides for gene analysis

Individual cells are prepared from histological tissue sections of routinely formalin-fixed and paraffin-embedded tissues using an ultraviolet laser micromanipulator. This technology, in combination with polymerase chain reaction (PCR)-based gene analysis, will enable researchers to routinely detect a variety of nucleic acid abnormalities underlying cancer, infection, and genetic disease with previously unknown sensitivity: at the single cell level. The utility of this technique is demonstrated by PCR amplification and sequencing of the E-cadherin gene, which codes for a homophilic cell-to-cell adhesion molecule, in early gastric carcinomas of the diffuse type of Lauren's classification. The main characteristics of the laser-assisted microdissection technique are high precision without contamination and easy application. The assignment of individual gene sequences to single cells will now provide a direct link between molecular biology on the one hand and histology and pathology on the other.     

Alkaline earth metal-based catalyst and process for selective hydrocarbon conversion to acetylene and carbon monoxide

A multi-step process that employs a Ni-modified mixed alkaline earth metal oxides (AEMO) has been developed for the selective conversion of hydrocarbons to C₂H₂ and CO. The initial process step is the catalytic decomposition of a gaseous hydrocarbon mixture at an elevated temperature (ca. 800 °C) over Ni/AEMO, generating H₂, trace CO, carbon (C), and trace alkaline earth metal carbide (MC₂). The Ni/AEMO/C/MC₂ material is then heated to consume the remaining carbon, generate more MC₂, and evolve CO. Then, Ni/AEMO/MC₂ is cooled and reacted with excess H₂O at a low temperature (20 < T (°C) < 80) to selectively generate C₂H₂ and Ni/AEM(OH)₂·zH₂O. In the final process step, Ni/AEM(OH)₂·zH₂O is decomposed to yield H₂O and Ni/AEMO, which is recycled within the process. The most advanced Ni/AEMO materials developed thus far exhibit intrinsic production capacities exceeding 2000 μmoles of C₂H₂ per gram of Ni/AEMO per process cycle.     

Supportive friendships moderate the association between stressful life events and sexual risk taking among African American adolescents.

Objective: This study examined whether uncontrollable stressful life events were associated with sexual risk taking among adolescents across a 1-year period, and whether supportive friendships modified associations. Design: Participants were 159 sexually active African American adolescents (57% male; mean age [SD] = 17.0 [1.5] years at baseline). Participants were recruited for in-person interviews through random digit dialing in one inner-city neighborhood characterized by high rates of poverty and crime relative to the surrounding city. Main Outcome Measures: Dependent variables included substance use before sexual activity and inconsistent condom use. Results: Among adolescents who reported low levels of supportive friendships, uncontrollable stressors were associated with greater levels of sexual risk taking over time. In contrast, uncontrollable stressors were not associated with sexual risk taking among adolescents who reported high social support from friends; risk taking was typically moderate to high among these adolescents. Conclusion: Different processes may explain sexual risk taking among adolescents with varying levels of social support from friends. Adolescents with low support may be prone to engagement in health risk behavior as a stress response, while adolescents with high support may engage in risk behavior primarily due to peer socialization of risk. (PsycINFO Database Record (c) 2010 APA, all rights reserved)