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S R Kain

Publications and source records attributed to S R Kain.

At least 37 records · Page 2Linked to original sources
PubMed1997

Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein.

The 2.1-A resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 --> Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore's excited state to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.

Amino Acid Sequence↗
PubMed1997

Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.

We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.

Escherichia coli↗
PubMed1997

Tracking and quantitation of retroviral-mediated transfer using a completely humanized, red-shifted green fluorescent protein gene.

We have developed murine retroviral vectors (RVs) containing an optimized green fluorescent protein (GFP) gene to study retroviral gene transfer and expression in living cells. We used the codon "humanized", "red-shifted" GFP gene, hGFP-S65T, a gain of function variant of the wild-type GFP from the jellyfish Aequorea victoria. We cloned the hGFP-S65T gene into the RV plasmid pLNCX (pLNChG65T). A stable amphotropic RV-producer cell line (VPC), designated LNChG65T VPC, was generated that exhibited bright fluorescence in greater than 95% of the cells. Human A375 melanoma cells and IGROV ovarian carcinoma cells transduced from LNCh-G65T VPC demonstrated high levels of fluorescence. The expression of a single integrated hGFP-S65T gene in eukaryotic cells provides a powerful tool to study gene transfer, expression and functional studies in vitro and in vivo.

Animals↗
PubMed1997

Early detection of apoptosis using a fluorescent conjugate of annexin V.

Apoptosis of mammalian cells is accompanied by various morphological changes including nuclear condensation, DNA fragmentation and cell surface changes. Methods developed over the past few years have focused on detection of DNA-associated changes that occur rather late in apoptosis. However, detection of apoptosis at early stages, before gross morphological changes, is critical for understanding the pathways of programmed cell death. In this report, we describe a rapid and reliable assay for detecting early stages of apoptosis. This assay is based on the observation that soon after initiating apoptosis, most mammalian cell types translocate phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be specifically detected by staining with fluorescein isothiocyanate (FITC)-labeled annexin V (annexin V-FITC), a protein with a strong, natural affinity for PS. Using this assay, we have detected apoptotic cells in culture, in real time, using fluorescence microscopy and flow cytometry. In combination with vital dye staining, the progressive stages of apoptosis were observed. PS redistribution occurs earlier than DNA-associated changes and membrane leakage. In addition, PS externalization occurs during apoptosis induced by a variety of stimuli. Therefore, the annexin V binding assay provides an excellent indicator for the early stages of apoptosis.

Annexin A5↗
PubMed1997

Quantification of gene expression with a secreted alkaline phosphatase reporter system.

The cDNA encoding secreted alkaline phosphatase (SEAP) is a useful tool for investigating the function of known or putative enhancer/promoter elements. SEAP has the unusual properties of extreme heat stability and resistance to the phosphatase inhibitor L-homoarginine. Therefore, endogenous alkaline phosphatase activity in transfected cells can be minimized by pretreatment of samples at 65 degrees C and incubation with the inhibitor. With the use of the chemiluminescent substrate CSPD, 10(-13) g of enzyme can be detected in culture medium, and the enzyme activity can be detected as early as 24 h after transfection. The chemiluminescence-based SEAP assay is about 10-fold more sensitive than similar assays using firefly luciferase as the reporter enzyme. The SEAP activity can also be assayed with a fluorescent substrate MUP, which provides sensitivity comparable to luciferase. Since the enzyme is secreted to culture medium, the enzyme assay can be performed on small samples of the culture supernatant. Because preparation of cell lysates is not required, assaying for SEAP activity is faster and more convenient than assaying for intracellular reporters. Furthermore, because the transfected cells are not disturbed by the sampling procedure, the same cultures can be repeatedly sampled for time-course studies or used for further investigations.

Alkaline Phosphatase↗
PubMed1996

Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein.

The green fluorescent protein (GFP) from Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of cells and organisms. Despite many early successes using this reporter, wild type GFP is suboptimal for most applications due to low fluorescence intensity when excited by blue light (488 nm), a significant lag in the development of fluorescence after protein synthesis, complex photoisomerization of the GFP chromophore and poor expression in many higher eukaryotes. To improve upon these qualities, we have combined a mutant of GFP with a significantly larger extinction coefficient for excitation at 488 nm with a re-engineered GFP gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yield an enhanced GFP which provides greater sensitivity in most systems.

Animals↗
PubMed1996

An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an important marker of gene expression. However, the sensitivity of wild-type GFP has been below that of standard reporter proteins, such as beta-galactosidase, which utilize enzymatic amplification. To improve the detection of GFP in transfected mammalian cells, we have constructed a unique GFP variant which contains chromophore mutations that make the protein 35 times brighter than wild-type GFP, and is codon-optimized for higher expression in mammalian cells. These changes in the GFP coding sequence provide an enhanced GFP (EGFP) that greatly increases the sensitivity of the reporter protein. We show that the EGFP expression vector delivered into mammalian cells gives rise to bright fluorescence that is readily detectable following a 16-24 hr transfection interval. Visual detection of transfected cells with EGFP appears to be more sensitive than equivalent measurements with beta-galactosidase catalyzed conversion of the X-gal substrate. We conclude that EGFP allows sensitive and convenient detection of gene transfer in mammalian cells.

Animals↗
PubMed1996

An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells.

We describe an acid phosphatase assay for determination of cell growth based on quantification of cytosolic acid phosphatase activity. The assay is based on the hydrolysis of the p-nitrophenyl phosphate by intracellular acid phosphatases in viable cells to produce p-nitrophenol. For all cell types examined, absorbance of p-nitrophenol at 405 nm is directly proportional to the cell number in the range of 10(3)-10(5) cells. The assay can quantify as few as 1000 cells per well in 96-well microtiter plates. The acid phosphatase assay was used to count various adherent and nonadherent cells, including human tumors, L6, and HT-2 cells. We also demonstrate the utility of this assay for analysis of growth factor and cytokine bioactivity on mammalian cells in culture. In comparison to [3H]thymidine incorporation, the acid phosphatase assay has similar sensitivity but a wider linear response range. The method also shows higher sensitivity and reproducibility in comparison to cell proliferation assays based on the reduction of tetrazolium salts. Because of the ease of use, sensitivity, and low cost, the acid phosphatase method is especially suited to applications where a large number of samples are assayed.

Acid Phosphatase↗
PubMed1996

Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant.

The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms. GFP emits bright green light (lambda max = 510 nm) when excited with ultraviolet (UV) or blue light (lambda max = 395 nm, minor peak at 470 nm). The chromophore in GFP is intrinsic to the primary structure of the protein, and fluorescence from GFP does not require additional gene products, substrates or other factors. GFP fluorescence is stable, species-independent and can be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry [Chalfie et al., Science 263 (1994) 802-805; Stearns, Curr. Biol. 5 (1995) 262-264]. The protein appears to undergo an autocatalytic reaction to create the fluorophore [Heim et al., Proc. Natl. Acad. Sci. USA 91 (1994) 12501-12504] in a process involving cyclization of a Tyr66 aa residue. Recently [Delagrave et al., Bio/Technology 13 (1995) 151-154], a combinatorial mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A. victoria GFP, yielding a number of different mutants with red-shifted fluorescence excitation spectra. One of these, RSGFP4, retains the characteristic green emission spectra (lambda max = 505 nm), but has a single excitation peak (lambda max = 490 nm). The fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP from the sea pansy, Renilla reniformis [Ward and Cormier, Photobiochem. Photobiol. 27 (1978) 389-396]. In the present study, we demonstrate by fluorescence microscopy that selective excitation of A. victoria GFP and RSGFP4 allows for spectral separation of each fluorescent signal, and provides the means to image these signals independently in a mixed population of bacteria or mammalian cells.

Amino Acid Sequence↗
PubMed1996

Transfection maximizer increases the efficiency of calcium phosphate transfections with mammalian cells.

A variety of methods have been described for introducing DNA into cultured mammalian cells. Among these, calcium phosphate (CaPO4)-mediated transfection has gained wide acceptance due to the procedure's low cost, simplicity and suitability for a wide range of cell types. However, this method is limited by relatively low transfection efficiencies and poor reproducibility. To improve the utility of CaPO4 transfections, we have identified a transfection enhancer (CalPhos Maximizer) that dramatically increases the efficiency of CaPO4 transfections with mammalian cells. The CalPhos Maximizer is a cellular structural protein that improved CaPO4 transfections with all mammalian cell lines tested and yielded greater than 70% transfection efficiency in BHK-21 cells. In the presence of the CalPhos Maximizer, transfection times were decreased to as little as 2 h with no loss in performance. Because of the high transfection efficiency provided by the CalPhos Maximizer transfection system, gene expression can be assayed following overnight incubations, rather than the typical 48-72-h intervals required by conventional transfection methods. Moreover, the Maximizer reagent is compatible with, and improves the performance of, commercially available as well as home-made CaPO4 transfection systems.

Animals↗
PubMed1995

Fast hybridization solution for the detection of immobilized nucleic acids.

We have developed a fast hybridization solution, termed ExpressHyb, for the rapid and sensitive detection of nucleic acids immobilized on membrane supports. This solution reduces typical hybridization times of 12-24 h to as little as 1 h while simultaneously increasing the sensitivity of detection in many applications. Using ExpressHyb, human beta-actin mRNA was detected on a human multiple tissue Northern (MTN) blot following a 30-min hybridization, with optimal detection occurring with a 1-h hybridization interval. The moderately abundant human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was detected using similar hybridization conditions and yielded improved signal-to-background characteristics relative to overnight hybridizations in conventional solutions. ExpressHyb can be used with either 32P- or digoxigenin-labeled probes and works effectively with both cDNA and oligonucleotide probes. For non-isotopic detection in particular, ExpressHyb reduces the nonspecific background commonly encountered with this technique. In cDNA library screening, ExpressHyb was found to both reduce the time required for effective hybridizations and to increase the number of positive colonies obtained relative to conventional overnight procedures. Taken together, these results illustrate the broad capability of ExpressHyb Hybridization Solution to improve nucleic acid detection in a variety of important techniques.

Actins↗
PubMed1995

Green fluorescent protein as a reporter of gene expression and protein localization.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is rapidly becoming an important reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (lambda max = 509 nm) when excited with UV or blue light (lambda max = 395 nm, minor peak at 470 nm). The fluorescence excitation and emission spectra of GFP are similar to those of fluorescein, and the conditions used to visualize this fluorophore are also suitable for GFP. Unlike other bioluminescent reporters, the chromophore in GFP is intrinsic to the primary structure of the protein, and GFP fluorescence does not require a substrate or cofactor. GFP fluorescence is stable, species-independent and can be monitored non-invasively in living cells and, in the case of transparent organisms, whole animals. Here we demonstrate GFP fluorescence in bacterial and mammalian cells and introduce our Living Colors line of GFP reporter vectors, GFP protein and anti-GFP antiserum. The reporter vectors for GFP include a promoterless GFP vector for monitoring the expression of cloned promoters/enhancers in mammalian cells and a series of six vectors for creating fusion protein to either the N or C terminus of GFP.

3T3 Cells↗
PubMed1994

Human multiple tissue western blots: a new immunological tool for the analysis of tissue-specific protein expression.

Human multiple tissue Western (MTW) blots are premade immunoblots prepared using proteins isolated from adult human tissue. The proteins are isolated from whole tissue homogenates under conditions designed to minimize proteolysis and to ensure maximal representation of tissue-specific proteins. Sodium dodecyl sulfate (SDS) solubilized proteins are fractionated by SDS polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes to generate blots ready for incubation with researcher-supplied antibodies. Each lane of an MTW blot contains an equivalent amount of total protein, allowing for the analysis of tissue-specific expression of a particular protein(s). The utility of MTW blots for Western blot applications was demonstrated by the detection of various cytoskeletal proteins and members of the annexin family of calcium-dependent, membrane-binding proteins. Several of these antigens were detected in separate cycles of antibody incubations using the same MTW blot. This approach is possible using a stripping procedure that allows the researcher to selectively remove both primary and secondary antibodies in a single incubation. The ease of multiple reprobings makes MTW blots both economical and convenient research tools for Western blot analyses of human tissue-specific proteins.

Adult↗
PubMed1993

Glucocorticoid-regulated trafficking of mouse mammary tumor virus proteins in permeabilized hepatoma cells. Requirements of intracellular membrane transport for maturation of the cytoplasmic phosphorylated polyprotein.

Glucocorticoids coincidentally regulate the localization of mouse mammary tumor virus (MMTV) glycoproteins and maturation of viral phosphoproteins in viral infected rat hepatoma cells. To test for a functional interaction between MMTV transmembrane glycoproteins and cytoplasmic phosphoproteins, the bacterial cytolysin streptolysin-O was utilized to selectively permeabilize the plasma membrane and reconstitute exocytic trafficking. Streptolysin-O-permeabilized M1.54 cells pretreated with glucocorticoids retained the capability for proteolytic processing, cell surface delivery, and externalization of MMTV glycoproteins as determined by immunoprecipitation and immunofluorescence microscopy. The efficient maturation of MMTV phosphoproteins indicated that these viral proteins are properly transported near or to the plasma membrane in permeabilized cells. These maturation events in semi-intact cells were dependent on the addition of cell cytosol and were specifically inhibited by the membrane impermeant GTP analog guanosine 5'3-O-(thio)triphosphate, an agent known to impede vesicular transport of membrane proteins, but which has not previously been shown to alter cytoplasmic protein maturation or transport. The addition of anti-MMTV antibodies directed against the cytoplasmic domain of the glycoprotein precursor to transport competent semi-intact M1.54 cells resulted in the dramatic inhibition of both MMTV glycoprotein and phosphoprotein maturation. These results were not obtained using either preimmune sera or antiserum specific for the luminal portion of the glycoprotein precursor. Our findings suggest that the functional interaction of cytosolic MMTV phosphoproteins with the cytoplasmic domain of the viral membrane glycoprotein is required for the efficient transport and processing of each class of proteins in glucocorticoid-treated cells and provides the first evidence for the involvement of vesicular transport in the delivery and maturation of cytoplasmic viral proteins at the plasma membrane or the pericellular region.

Animals↗
PubMed1993

Trafficking of wild-type and an endoproteolytic-site mutant of the mouse mammary tumor virus glycoprotein.

We have utilized a mouse mammary tumor virus (MMTV) glycoprotein gene containing a mutation in the endoproteolytic cleavage site to investigate the biological significance of processing, the structural requirements for and the events involved in the proteolytic maturation of MMTV. Using oligonucleotide-directed mutagenesis, the endoproteolytic cleavage site within the MMTV glycoprotein was mutated from Arg-Ala-Lys-Arg to Arg-Ala-Asn-Gln and both the wild-type and mutated genes were transfected and expressed in HTC rat hepatoma cells. Indirect immunofluorescence, steady state radiolabeling and pulse-chase kinetic experiments demonstrated that this mutated glycoprotein was transported to the cell surface with the same efficiency as the wild-type maturation products; however, proteolytic cleavage and fusogenic activity were almost completely abolished. Consistent with the lack of cleavage, the endoglycosidase H-resistant precursor, gp78, accumulated on the cell surface and in the extracellular environment. When HTC cells expressing the wild-type MMTV glycoprotein were treated with the Golgi mannosidase I and II inhibitors, deoxymannojirimycin and swainsonine respectively, the resulting endogycosidase H-sensitive glycoproteins were processed efficiently. Taken together, these results suggest that proteolytic processing of the MMTV glycoprotein most likely occurs in the trans Golgi or at a later step in the exocytic pathway and occurs after the formation of an endoglycosidase H-resistant, terminally sialylated intermediate. Moreover, the acquisition of endoglycosidase H-resistant oligosaccharides is not a prerequisite for recognition by the cellular proteases to generate the viral maturation products. Our evidence also suggests that the processing of the MMTV envelope glycoprotein is required for the functional exposure of the fusion domain which is involved in viral infectivity.

Animals↗