Method for monitoring of the protein amino group changes during carbonylation

Objectives

Carbonylation of the protein amino, guanidino and thiol groups is one of the important causes of vascular complications in diabetes. We developed a simple spectrophotometric method for monitoring of the changes in the protein amino group contents during carbonylation.

Design and methods

The method is based on the reaction of amino group with p-benzoquinone in the slightly alkaline media. It was applied during carbonylation in vitro with methylglyoxal and in vivo in 13 patients with type 2 diabetes and 20 healthy persons.

Results

The method is simple, fast, precise (RSD in the range of 1.2–1.8%) and accurate (recovery about 100%). The content of amino groups in human serum albumin isolated from diabetics was significantly lower (p < 0.01) in comparison with a control group.

Conclusion

The method developed is suitable for quantification of protein amino groups during in vitro carbonylation as well as for clinical practice.     

Enucleation of feeder cells and egg cells with psoralens

The cell nucleus must be inactivated or destroyed in order to generate feeder layers for cultured cells or to prepare recipient egg cells for nuclear transfer. Existing enucleation techniques are either cumbersome or employ toxic chemicals. Here we report a new method to enucleate cells by treatment with a psoralen and long‐wave ultraviolet light. The technique is >90% efficient and causes little cytoplasmic damage to the treated cell. We have used psoralen treatment to enucleate a wide variety of cells, including eggs, sperm, HeLa cells, and fibroblasts. Colonies of human embryonic stem cells (hESCs) and human keratinocyte precursors grown on psoralen‐treated feeders are indistinguishable from those grown on gamma‐irradiated or mitomycin C‐treated cells. Psoralen enucleation provides a rapid, simple, and non‐toxic method to generate feeder cells. The technique is also useful for nuclear transfer studies in species with large eggs whose cleavage divisions are not regulated by cell‐cycle checkpoints. Developmental Dynamics 238:2614–2621, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of residual ischemia in the occluded coronary artery irrigation area using myocardial perfusion scintigraphy

BACKGROUND: Inspite the indisputable significance of coronarography, the implications of a revealed stenosis--and how close it is to occlusion could vary in regard to its physiological importance. Myocardial perfusion scintigraphy (MPS) possiblity to prove and objectivise the presence of ischemia and myocardial viability within an occlusion found coronarographically is especially significant since it makes possible to the clinitian to choose an adequate therapy. CASE REPORT: We reported a 43-year-old male patient who had been hospitalized to another institution due to acute myocardial infarction (AMI) of posterolateral localisation. Following the acute AMI stage the ergometric test per Bruce protocole was performed, negative to ischemic heart disease, while multislice computed tomography showed no significant changes on the coronary arteries. The performed one-day-protocole MPS showed a massive area of residual ischemia within myocardial infarction (MI) type culprit lesion of the posterolateral zone starting from the subapical level to the basal cross-section. According to the MPS findings coronarography was indicated due to a revascularisation assessment. The performed coronarography revealed an occlusion of the circumflex coronary artery (CCA) right after the division of obtuse branch (OB) that presented discretely in the distal parts from the homo- and heterocolaterals. The distal portion of CCA presented discretely out off the right coronary artery (RCA). Echocardiography confirmed an ejection fraction of 50% with hypokinesia of inferior and posterior walls, as well as the septum, showing a mild reduction of the general contractility. Flows through confluences were well. A month after MI a percutane coronary intervention (PCI) was performed with the implantation of a drug-releasing stent (Taxus). Early after PCI (within two weeks) a control MPS was done to evaluate the effects of the therapy giving the normal findings of myocardial perfusion. CONCLUSION. Determination and identificantion of vivid but ischemic myocard of culprit lesion type in the occluded artery irrigation zone enable making choice of the best therapy for a patient.     

Chronic isolation stress compromises JNK/c-Jun signaling in rat brain

The c-Jun NH2-terminal kinases (JNKs) are important stress-responsive kinases. They regulate cellular activities by sequential phosphorylation and activation through a mitogen-activated protein kinase cascade, whereas JNKs activation is altered in response to various stressors. In the present study, we used immunoblotting to assess the effect of 21?day of social isolation as the chronic stressor, either sole and in combination with 2?h of acute immobilization or cold (4°C) stress on circulating corticosterone level and phosphorylation status of p46 (phospho-p46/total p46) and p54 (phospho-p54/total p54) JNK isoforms in the cytosolic and nuclear fraction of the prefrontal cortex and hippocampus of male Wistar rats. Also, the phosphorylation status of JNK nuclear down-stream target c-Jun (p–c-Jun/c-Jun) on Ser63 was examined. Both acute stressors with elevated CORT levels led to increased phosphorylation status of cytosolic p54 JNK isoforms but not p46 JNK isoforms only in the hippocampus and no change in phosphorylation status of c-jun in both brain regions. Chronic isolation with unaltered CORT level and reduced responsiveness to novel acute stressors, led to unchanged or reduced phosphorylation status of p46 and p54 JNK isoforms in both fractions and both brain regions, whereas the decrease of c-Jun phosphorylation status was found only in the prefrontal cortex. Our results suggest that compromised JNKs activation following chronic isolation may lead to interruption of JNK signaling, which could be related with neuropsychiatric disorders such as depression or long-lasting neuronal remodeling.     

The Dipolar ElectroCARdioTOpographic (DECARTO)-like method for graphic presentation of location and extent of area at risk estimated from ST-segment deviations in patients with acute myocardial infarction

A graphic method was developed for presentation of the location and extent of the myocardium at risk in patients with acute myocardial infarction (AMI). This method is based on a mathematical processing of ST-segment deviations of standard 12-lead electrocardiogram following the concept of Titomir and Ruttkay-Nedecky in their dipolar electrocardiotopographic method. The center of the location of the area at risk is given by the spatial orientation of the resultant spatial ST vector, and the extent of the area at risk is derived from the Aldrich score. The areas at risk are projected on a spherical image surface, on which a texture of the anatomical quadrants of the ventricular surface and its coronary artery supply are projected. The method was tested in 10 patients with AMI with single-vessel disease, including 6 patients with an occlusion in the proximal left anterior descending coronary artery (LAD), 3 patients with an occlusion in the right coronary artery, and one patient with occlusion in the left circumflex coronary artery. The estimated areas at risk were compared with myocardial perfusion single photon emission computed tomography. Eight (80%) patients of 10 were correctly localized according to the Aldrich decision rules for the location of AMI. The areas at risk in patients with LAD occlusion correctly localized by the Aldrich score were situated in the anteroseptal and anterosuperior quadrants. In the inferior AMI group, the area at risk was localized in the posterolateral and inferior quadrants. The visual comparison with myocardial perfusion single photon emission computed tomography (SPECT) showed best agreement in patients with LAD involvement. The initial testing showed that this method allows a graphic presentation of estimated area at risk using clinically defined diagnostic rules. The area at risk can be displayed in images that are familiar for clinicians and can be compared with or superimposed on results of other imaging methods used in cardiology.