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Biomedical subjects

Srinagesh V Koushik

Publications and source records attributed to Srinagesh V Koushik.

8 recordsLinked to original sources
PubMed2006

Cerulean, Venus, and VenusY67C FRET reference standards.

Förster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.

Cell Line↗
PubMed2006

Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells.

Measurement of fluorescence resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores in living cells using the three-filter cube approach requires the determination of two constants: 1), the ratio of sensitized acceptor emission to donor fluorescence quenching (G factor) and 2), the ratio of donor/acceptor fluorescence intensity for equimolar concentrations in the absence of FRET (k factor). We have developed a method to determine G and k that utilizes two donor-acceptor fusion proteins with differing FRET efficiencies-the value of which need not be known. We validated the method by measuring the FRET efficiency and concentration ratio of the fluorescent proteins Cerulean and Venus in mammalian cells expressing a series of fusion proteins with varying stoichiometries. The method greatly simplifies quantitative FRET measurement in living cells as it does not require cell fixation, acceptor photobleaching, protein purification, or specialized equipment for determining fluorescence spectra or lifetime.

Algorithms↗
PubMed2006

Fanciful FRET.

The validity of experiments based on Förster resonance energy transfer (FRET), an imaging technique widely used to measure protein-protein interactions in living cells, critically depends on the accurate and precise measurement of FRET efficiency. The use of FRET standards to determine FRET efficiency, and a consideration of such factors as how the abundance of FRET acceptors and the stoichiometry of donors and acceptors in a molecular complex can affect measured FRET efficiency, will enhance the usefulness with which FRET experiments can be interpreted.

Algorithms↗
PubMed2005

Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer.

Protein labeling with green fluorescent protein derivatives has become an invaluable tool in cell biology. Protein quantification, however, is difficult when cells express constructs with overlapping fluorescent emissions. Under these conditions, signal separation using emission filters is inherently inefficient. Spectral imaging solves this problem by recording emission spectra directly. Unfortunately, linear unmixing, the algorithm used for quantifying individual fluorophores from emission spectra, fails when resonance energy transfer (RET) is present. We therefore sought to develop an unmixing algorithm that incorporates RET. An equation for spectral emission incorporating RET was derived and an assay based on this formalism, spectral RET (sRET), was developed. Standards with defined RET efficiencies and with known Cerulean/Venus ratios were constructed and used to test sRET. We demonstrate that sRET analysis is a comprehensive, photon-efficient method for imaging RET efficiencies and accurately determines donor and acceptor concentrations in living cells.

Algorithms↗
PubMed2003

What cardiovascular defect does my prenatal mouse mutant have, and why?

Since the advent of mouse targeted mutations, gene traps, an escalating use of a variety of complex transgenic manipulations, and large-scale chemical mutagenesis projects yielding many mutants with cardiovascular defects, it has become increasingly evident that defects within the heart and vascular system are largely responsible for the observed in utero lethality of the embryo and early fetus. If a transgenically altered embryo survives implantation but fails to be born, it usually indicates that there is some form of lethal cardiovascular defect present. A number of embryonic organ and body systems, including the central nervous system, gut, lungs, urogenital system, and musculoskeletal system appear to have little or no survival value in utero (Copp, 1995). Cardiovascular abnormalities include the failure to establish an adequate yolk-sac vascular circulation, which results in early lethality (E8.5-10.5); poor cardiac function (E9.0-birth); failure to undergo correct looping and chamber formation of the primitive heart tube (E9.0-11.0); improper septation, including division of the common ventricle and atria and the establishment of a divided outflow tract (E11.0-13.0); inadequate establishment of the cardiac conduction system (E12.0-birth); and the failure of the in utero cardiovascular system to adapt to adult life (birth) and close the interatrial and aorta-pulmonary trunk shunts that are required for normal fetal life. Importantly, the developmental timing of lethality is usually a good indicator of both the type of the cardiovascular defect present and may also suggest the possible underlying cause/s. The purpose of this review is both to review the literature and to provide a beginner's guide for analysing cardiovascular defects in mouse mutants.

Animals↗
PubMed2002

Cardiac-specific expression and hypertrophic upregulation of the feline Na(+)-Ca(2+) exchanger gene H1-promoter in a transgenic mouse model.

The NCX1 gene contains three promoters (H1, K1, and Br1), and as a result of alternative promoter usage and alternative splicing, there are multiple tissue-specific variants of the Na(+)-Ca(2+) exchanger. We have proposed that for NCX1, the H1 promoter regulates expression in the heart, the K1 promoter regulates expression in the kidney, and the Br1 promoter regulates expression in the brain as well as low-level ubiquitous expression. Here, using a transgenic mouse model, we test the role of the DNA region including -1831 to 67 bp of intron 1, encompassing exon H1 of the feline NCX1 gene (NCX1H1). The NCX1H1 promoter was sufficient for driving the normal spatiotemporal pattern of NCX1 expression in cardiac development. The luciferase reporter gene was expressed in a heart-restricted pattern both in early embryos (embryonic days 8 to 14) and in later embryos (after embryonic day 14), when NCX1 is also expressed in other tissues. In the adult, no luciferase activity was detected in the kidney, liver, spleen, uterus, or skeletal muscle; minimal activity was detected in the brain; and very high levels of luciferase expression were detected in the heart. Transverse aortic constriction-operated mice showed significantly increased left ventricular mass after 7 days. In addition, there was a 2-fold upregulation of NCX1H1 promoter activity in the left ventricle in animals after 7 days of pressure overload compared with both control and sham-operated animals. This work demonstrates that the NCX1H1 promoter directs cardiac-specific expression of the exchanger in both the embryo and adult and is also sufficient for the upregulation of NCX1 in response to pressure overload.

Animals↗
PubMed2002

Role of sodium-calcium exchanger (Ncx1) in embryonic heart development: a transgenic rescue?

Na(+)/Ca(2+) exchanger (Ncx-1) is highly expressed in cardiomyocytes, is thought to be required to maintain a low intracellular Ca(2+) concentration, and may play a role in excitation-contraction coupling. Significantly, targeted deletion of Ncx-1 results in Ncx1-null embryos that do not have a spontaneously beating heart and die in utero. Ultrastructural analysis revealed gross anomalies in the Ncx1-null contractile apparatus, but physiologic analysis showed normal field-stimulated Ca(2+) transients, suggesting that Ncx-1 function may not be critical for Ca(2+) extrusion from the cytosol as previously thought. Using caffeine to empty the intracellular Ca(2+) stores, we show that the sarcoplasmic reticulum is not fully functional within the 9.5-dpc mouse heart, indicating that the sarcoplasmic reticulum is unlikely to account for the unexpected maintenance of intracellular Ca(2+) homeostasis. Using the Ncx1-lacZ reporter, our data indicate restricted expression patterns of Ncx1 and that Ncx1 is highly expressed within the conduction system, suggesting Ncx1 may be required for spontaneous pacemaking activity. To test this hypothesis, we used transgenic mice overexpressing one of the two known adult Ncx1 isoforms under the control of the cardiac-specific a-myosin heavy chain promoter to restore Ncx1 expression within the Ncx1-null hearts. Results indicate that the transgenic re-expression of one Ncx1 isoform was unable to rescue the lethal null mutant phenotype. Furthermore, our in situ results indicate that both known adult Ncx1 isoforms are coexpressed within the embryonic heart, suggesting that effective transgenic rescue may require the presence of both isoforms within the developing heart.

Animals↗