Biomedical subjects

Sriram Srinivasan

Publications and source records attributed to Sriram Srinivasan.

5 recordsLinked to original sources

PubMed2004

High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification.

We report further development of a novel recombinant protein expression system based on the Gram-negative bacterium, Ralstonia eutropha. In this study, we were able to express soluble, active, organophosphohydrolase (OPH), a protein that is prone to inclusion body formation in Escherichia coli, at titers greater than 10 g/L in high cell density fermentation. This represents a titer that is approximately 100-fold greater than titers previously reported in E. coli for this enzyme. R. eutropha strains expressing OPH were generated in two cloning steps. First, the T7 RNA polymerase gene was placed under the control of the strong, inducible phaP promoter and integrated into the phaP locus of R. eutropha NCIMB 40124. Second, a single copy of the oph gene under control of the T7 promoter was randomly integrated into the chromosome using a transposon cloning vector.

Cloning, Molecular↗

PubMed2003

Production of recombinant proteins using multiple-copy gene integration in high-cell-density fermentations of Ralstonia eutropha.

We have previously reported the development of a novel protein expression system based on Ralstonia eutropha. In this study we report on the influence of gene copynumber on recombinant protein expression in R. eutropha. We compare recombinant gene stability and expression levels of chromosomal integration with a plasmid-based expression system. Single, double, and triple copies of a gene encoding organophosphohydrolase (OPH), an enzyme prone to inclusion-body formation in E. coli, were integrated into the R. eutropha chromosome. A linear increase between the concentration of soluble, active OPH and gene copynumber was found. Using a triple-copy integrant, we were able to produce approximately 4.3 g/L of OPH in a high-cell-density fermentation. This represents the highest titer reported to date for this enzyme, and is approximately 30 times greater than expression levels reported in E. coli.

Aryldialkylphosphatase↗

PubMed2003

Cardiac resynchronization therapy improves heart rate variability in patients with symptomatic heart failure.

BACKGROUND: Cardiac resynchronization therapy (CRT) using biventricular pacing improves symptoms and functional capacity in patients with moderate to severe heart failure. The present study examined whether an improvement in ventricular performance from resynchronization therapy changes the autonomic control of heart rate. METHODS AND RESULTS: Heart rate variability (HRV) was examined in 50 patients implanted with the InSync biventricular pacing system who were randomized to therapy-on (n=25) or therapy-off (n=25). HRV was computed as the standard deviation of the atrial cycle length sensed from the system over 2 months of continuous monitoring. HRV was compared between CRT-on and CRT-off groups. HRV was higher in patients randomized to CRT-on compared with CRT-off (148+/-47 ms for CRT-on versus 118+/-45 ms for CRT-off; P=0.02), despite the lack of difference in mean atrial cycle length (844+/-129 ms for CRT-on versus 851+/-110 ms for CRT-off; P=0.82). Changes in plasma catecholamines were not different between the CRT-on and CRT-off groups from baseline to the 3-month follow-up. CONCLUSIONS: Improvement in ventricular performance from CRT shifts cardiac autonomic balance toward a more favorable profile that is less dependent on sympathetic activation.

Adrenergic beta-Antagonists↗

PubMed2002

Estimation of noise level and signal to noise ratio of laplacian electrocardiogram during ventricular depolarization and repolarization.

Body surface Laplacian ECG (LECG) has demonstrated its enhanced capability to localize cardiac electrical sources closest to the recording site. The aim of the present study was to evaluate the noise level and signal to noise ratio (SNR) in the LECG as compared to the potential ECG (PECG). Such evaluation is important to determine the applicability of the LECG to localizing and imaging of cardiac electrical activity in an experimental setting. Experimental studies were conducted in six healthy men. A 150-channel PECG was recorded from the anterolateral chest and the LECG was estimated using the finite difference algorithm. The noise level in the PECG and LECG was evaluated using multiple estimation protocols. The signal level during ventricular depolarization and repolarization was also estimated, and the corresponding SNR was calculated. Different filtering techniques were examined to evaluate their effects on the noise level and SNR of the LECG and PECG. The experimental results indicate that with basic signal processing techniques (baseline adjustment, three-point moving average filter, and Wiener spatial filter), the SNR of the LECG is about 30-40% of that of the PECG. Furthermore, the SNR estimated during ventricular depolarization is about three times that obtained during ventricular repolarization for the PECG and LECG. The present study indicates that the LECG derived from the PECG using a local finite difference estimation procedure has satisfactory SNR during the periods of ventricular depolarization and repolarization, and suggests the feasibility of estimating the LECG from the recorded PECG in human subjects in an experimental setting.

Body Surface Potential Mapping↗

PubMed2002

A novel high-cell-density protein expression system based on Ralstonia eutropha.

We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli.

Cupriavidus necator↗