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Biomedical subjects

S R Kain

Publications and source records attributed to S R Kain.

At least 19 recordsLinked to original sources
PubMed1999

Characterization of NFkappaB activation by detection of green fluorescent protein-tagged IkappaB degradation in living cells.

Activation of the transcription factor NFkappaB requires rapid degradation of its inhibitor, IkappaBalpha. To facilitate the study of IkappaBalpha degradation, we fused IkappaBalpha protein to enhanced green fluorescent protein to construct IkappaBalpha-enhanced green fluorescent protein (IG). We demonstrated by both flow cytometry and Western blot analysis that the half-life of IG in the presence of human tumor necrosis factor (TNF) alpha is approximately 5 min, which is similar to the half-life of native IkappaBalpha. The degradation coincided with NFkappaB translocation from the cytoplasm to the nucleus and NFkappaB-mediated induction of transcription. Phorbol 12-myristate 13-acetate (PMA), but not forskolin, also induces degradation of IG fusion protein. The half-life of IG in the presence of PMA is approximately 15 min, longer than when induced with TNFalpha. Co-treatment with TNFalpha and PMA did not result in a synergistic effect on IG degradation, although they stimulate different kinases in two different signaling pathways. Degradation of IG was inhibited by mutations at serine residues 32 and 36, which are the target sites of the phosphorylation modification that initiates degradation of IkappaBalpha. We also demonstrated that basal degradation of IG in the presence of cycloheximide is inhibited by such mutations, suggesting that basal degradation of IkappaBalpha also requires phosphorylation as the signal for degradation. Finally, we showed that the rate of TNFalpha-induced degradation of IG remains almost constant throughout the cell cycle, except at the mitotic phase, in which IG degrades more slowly.

DNA-Binding Proteins↗
PubMed1998

Generation of destabilized green fluorescent protein as a transcription reporter.

The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFkappaB binding sequences and monitoring tumor necrosis factor alpha-mediated NFkappaB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFkappaB translocation to the nucleus.

Amino Acid Sequence↗
PubMed1998

Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of systems. Applications using GFP reporters have expanded greatly due to the availability of mutants with altered spectral properties, including several blue emission variants, all of which contain the single point mutation Tyr-66 to His in the chromophore region of the protein. However, previously described "BFP" reporters have limited utility, primarily due to relatively dim fluorescence and low expression levels attained in higher eukaryotes with such variants. To improve upon these qualities, we have combined a blue emission mutant of GFP containing four point mutations (Phe-64 to Leu, Ser-65 to Thr, Tyr-66 to His, and Tyr-145 to Phe) with a synthetic gene sequence containing codons preferentially found in highly expressed human proteins. These mutations were chosen to optimize expression of properly folded fluorescent protein in mammalian cells cultured at 37 degreesC and to maximize signal intensity. The combination of improved fluorescence and higher expression levels yield an enhanced blue fluorescent protein that provides greater sensitivity and is suitable for dual color detection with green-emitting fluorophores.

Fluorescence↗
PubMed1998

Dual-color flow cytometric detection of fluorescent proteins using single-laser (488-nm) excitation.

The ability to analyze independently the expression of multiple reporter gene constructs within single cells is a potentially powerful application of flow cytometry. In this paper, we explore the simultaneous detection of two variants of the reporter molecule, green fluorescent protein (GFP) that both fluoresce when excited with 488-nm light. One of these, enhanced GFP (EGFP) (excitation max. 490 nm; > 90% efficiency at 488 nm), has been widely used for studies that involve flow cytometric detection of reporter gene expression. As a partner for EGFP, we employed a recently described variant termed enhanced yellow fluorescent protein (EYFP) (excitation max. 513 nm; approximately 35% efficiency at 488 nm). Using 488-nm excitation, EYFP fluorescence could be readily detected following expression of the gene in murine fibroblasts and this signal was comparable in intensity to that obtained from EGFP. Importantly, we describe an optical filter configuration that permits the fluorescence signals from both proteins to be distinguished by flow cytometry, despite their similar emission maxima. This filter configuration employed a 510/20-nm bandpass filter for EGFP detection, a 550/30-nm bandpass filter for EYFP detection, and a 525-nm short-pass dichroic mirror to separate the two signals. With these filters, expression of either reporter protein could be detected, alone or in combination, within a mixed population of cells over a broad range of signal intensities.

3T3 Cells↗
PubMed1998

Caspase inhibition prevents staurosporine-induced apoptosis in CHO-K1 cells.

Apoptosis is a distinct form of programmed cell death that plays an important role in many biological processes. Although the phenotypes of apoptotic cells are well documented, little is known of the central mechanism leading to programmed cell death. Over the past few years, a number of ICE/CED-3 family proteases (also termed caspases) have been discovered and implicated as the common effectors of apoptosis. In this report, we demonstrate that induction of apoptosis in CHO-K1 cells by staurosporine, a broad spectrum inhibitor of protein kinases, results in an increase in DEVD-dependent protease activity. These events were followed by nuclear DNA fragmentation and cell death. Inhibition of the DEVD-cleaving activity by a synthetic tetrapeptide inhibitor DEVD-CHO, blocked staurosporine-induced downstream apoptotic phenotypes, such as morphological characteristics and DNA fragmentation. These results suggest that staurosporine-induced apoptosis in CHO-K1 cells is mediated through the CPP32/caspase-3-like cysteine proteases.

Journal Article↗
PubMed1997

Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence.

The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a widely used marker for gene expression and protein localization studies. Dissection of the structure of the protein would be expected to shed light on its potential applications to other fields such as the detection of protease activity. Using deletion analysis, we have defined the minimal domain in GFP required for fluorescence to amino acids 7-229. This domain starts at the middle of the first small alpha helix at the N terminus of GFP and ends immediately following the last beta sheet. Studies of the amino acids at both termini of the minimal domain revealed that positions 6 and 7 at the N terminus are Glu-specific. Change of the Glu residues to other amino acids results in reduction of GFP fluorescence. Position 229 at the C terminus of GFP, however, is nonspecific: the Ile can be replaced with other amino acids with no measurable loss of fluorescence. A total of only 15 terminal amino acids can be deleted from GFP without disrupting fluorescence, consistent with findings of a previous study of GFP crystal structure (Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., Remington, S. J. (1996) Science 273, 1392-1395 and Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nat. Biotechnol. 14, 1246-1251) that a tightly packed structure exists in the protein. We also generated internal deletions within the loop regions of GFP according to its crystal structure and found that all such deletions eliminated GFP fluorescence.

Animals↗
PubMed1997

Fluorometric and colorimetric detection of caspase activity associated with apoptosis.

The caspase (ICE/CED-3) family of proteases has been implicated to play a crucial role in apoptosis. However, the mechanisms by which caspase activity mediates apoptosis are not fully understood. Progress in this area has been limited due to the lack of a convenient and reliable system to quantify these protease activities. In this report, we describe a quantitative assay for the activity of caspase-3, a member of the caspase family thought to mediate apoptosis in most mammalian cell types. This assay utilizes a synthetic tetrapeptide, Asp-Glu-Val-Asp (DEVD), labeled with either a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC), or a colorimetric molecule, p-nitroanilide (pNA) as substrates. DEVD-dependent protease activity is assessed by detection of the free AFC or pNA cleaved from the substrates. We demonstrate the utility of the assay for rapid quantification of caspase-3 activity in the onset of apoptosis. Using the assay, we show that apoptosis induced in 32D cells under various conditions is associated with an increase in the DEVD-dependent protease activity. These studies suggest that induction of the DEVD-dependent protease activity is an indicator of apoptosis and demonstrate the utility of the assays for assessment of the role of caspase-family proteases in apoptotic cell progression.

Amino Acid Sequence↗
PubMed1997

A cationic lipid for rapid and efficient delivery of plasmid DNA into mammalian cells.

Cationic lipids are widely used for gene transfer into cultured eukaryotic cells. However, lipids with potent transfection activity are often associated with high levels of cytotoxicity, and also require serum-free conditions for optimal performance. These characteristics in many cases result in unsatisfactory transfection efficiency. In this report, we describe a new cationic amphiphile, N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (Amidine). Amidine requires only 1-2 hour incubation intervals to produce maximal transfection efficiency, and can transfect cells in the presence of serum. Such characteristics significantly minimize cytotoxicity, and also provide time flexibility for researchers. We routinely obtain over 80% transfection efficiency as evidenced by use of an enhanced green fluorescence protein (EGFP) as the reporter. These studies demonstrate the utility of Amidine for rapid and efficient transfection of mammalian cells.

Animals↗