Acute volume overload elevates T-wave alternans magnitude.

The objective of this study was to determine whether acute volume loading elevates T-wave alternans (TWA) in dogs with structurally normal hearts. TWA predicts sudden cardiac arrest in patients with left ventricular dysfunction and congestive heart failure. However, volume load and ventricular stretch may themselves precipitate arrhythmias. It is unclear to what extent volume load causes TWA. In six male mongrel dogs [25.8 kg (SD 4.2)] under general anesthesia, we measured TWA during progressive atrial pacing to 160 beats/min. Pacing was performed at baseline, at the midpoint and peak of a saline infusion designed to induce acute CHF, and then during diuresis. Dog 1 was hypothermic throughout the protocol and excluded from analysis. For dogs 2-6, 102 ml/kg (SD 30) were infused over 315 min (SD 50), causing pulmonary capillary wedge pressure to rise from 9.6 (SD 3.5) to 21.2 mmHg (SD 1.6) (P < 0.01), and heart rate variability to fall (P < 0.01). TWA magnitude (Valt) rose in all dogs with volume load (P < 0.001). Compared with baseline, TWA at peak infusion had higher magnitude [Valt 3.4 (SD 1.95) vs. 0.5 muV (SD 0.35); P = 0.011] and occurred at lower heart rates [128 (SD 6) vs. 151 beats/min (SD 12); P = 0.008]. Net volume load was linearly related to Valt (P < 0.01), with each 10 ml/kg net volume load increasing Valt by 0.23 muV. Acute volume overload elevates TWA in normal canine hearts. Although dramatic, however, this effect may contribute clinically to abnormal TWA only in patients with marked volume overload. Future studies should examine the interaction of fluid overload, myocardial disease, and arrhythmia susceptibility.     

Automated laser scanning cytometry: a powerful tool for multi-parameter analysis of drug-induced apoptosis.

BACKGROUND: Simultaneous analysis of multiple intracellular events is critical for assessing the effect of biological response modifiers, including the efficacy of chemotherapy. Here we used the automated laser scanning cytometry (LSC) for multi-parameter analysis of drug-induced tumor cell apoptosis. MATERIALS: Using 2-mercaptopyridine-N-oxide-hydrate sodium salt, or the commonly used chemotherapeutic agents etoposide and camptothecin, we performed simultaneous analyses of apoptosis-related morphological features as well as fluorescence-based biochemical changes in a 96-well format. RESULTS: We demonstrate the scope of LSC as a platform for comparing multiple variables between different cell populations, distinguishing unique events at a single cell level within a sample population, and enabling simultaneous screenings in a single assay at multiple dosages and time-points. CONCLUSION: These data underscore the power of LSC for simultaneous multi-parameter analysis, which could have implications for screening or assessing the efficacy of drug responses in heterogeneous cell populations and at the single cell level.     

BRET-based method for detection of specific RNA species

RNA detection and quantitation is a common necessity in modern molecular biology research. Most methods, however, are complex and/or time-intensive. Presented here is a BRET (bioluminescene resonance energy transfer)-based method that can accomplish the task of RNA identification quickly and easily. By conjugating BRET enzymes to two different oligonucleotides that are complementary to the same target sequence, probes were developed that could detect RNA using a solution-based assay. This assay was optimized for spacer length between the binding sites (found to be 10 nucleotides), and sensitivity was determined to be 1 microg for a specific species of RNA within a mixed population. Specificity of the assay was assessed using in vitro transcribed cRNA and found to be statistically siginificant ( p = 3.11 x 10 (-6), ANOVA, multiple range test). This assay represents a possibility for a less technically demanding, streamlined alternative to canonical RNA assays.     

DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet

Using a bisubstituted caspase-3 target sequence: aspartate-glutamate-valine-aspartate, (z-DEVD)2 peptide derivative of the fluorophore, cresyl violet, we have obtained a cell permeant, fluorogenic, caspase substrate capable of detecting the site-specific presence of functionally active, caspase-3 and caspase-7 up-regulation within intact apoptotic cells. Addition of this substrate to induced and noninduced cell culture populations allows for the rapid site-specific detection of caspase up-regulation without the requirement for a wash step. We demonstrate here the use of (z-DEVD)2-cresyl violet substrate for the detection of apoptosis induction in Jurkat, THP-1, and MCF-7 cells using fluorescence microscopy and 96-well fluorescence plate reader analysis. Intracellular up-regulated DEVDase activity, which was clearly visible by fluorescence microscopy and 96-well fluorescence plate reader measurements, showed greater than 6-fold increases in fluorescence output in induced versus noninduced Jurkat cell samples. A simple fluorogenic substrate conversion method is demonstrated here for detecting apoptosis induction within intact living cells.