Biomedical subjects

D W Piston

Publications and source records attributed to D W Piston.

30 records · Page 2Linked to original sources

PubMed1997

Cell-specific expression and regulation of a glucokinase gene locus transgene.

Transgenic mice containing one or more extra copies of the entire glucokinase (GK) gene locus were generated and characterized. The GK transgene, an 83-kilobase pair mouse genomic DNA fragment containing both promoter regions, was expressed and regulated in a cell-specific manner, and rescued GK null lethality when crossed into mice bearing a targeted mutation of the endogenous GK gene. Livers from the transgenic mice had elevated GK mRNA, protein, and activity levels, compared with controls, and the transgene was regulated in liver by dietary manipulations. The amount of GK immunoreactivity in hepatocyte nuclei, where GK binds to the GK regulatory protein, was also increased. Pancreatic islets displayed increased GK immunoreactivity and NAD(P)H responses to glucose, but only when isolated and cultured in 20 mM glucose, as a result of the hypoglycemic phenotype of these mice (Niswender, K. D., Shiota, M., Postic, C., Cherrington, A. D., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22604-22609). Together, these results indicate that the region of the gene from -55 to +28 kilobase pairs (relative to the liver GK transcription start site) contains all the regulatory sequences necessary for expression of both GK isoforms, thereby placing an upper limit on the size of the GK gene locus.

Animals↗

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

Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development.

Bone Morphogenetic Proteins (BMPs) play crucial roles in a variety of developmental processes, but their functions during early vertebrate brain development are largely unknown. To investigate this problem, we have compared by in situ hybridization the expression of five Bmp genes belonging to the Drosophila Decapentaplegic (Bmp2 and Bmp4) and 60A subgroups (Bmp5, Bmp6 and Bmp7). Striking co-expression of these Bmps is observed within the dorsomedial telencephalon, coincident with a future site of choroid plexus development. Bmp co-expression overlaps that of Msx1 and Hfh4, and is complementary to that of Bf1. The domain of Bmp co-expression is also associated with limited growth of the neuroectoderm, as revealed by morphological observation, reduced cell proliferation, and increased local programmed cell death. In vitro experiments using explants from the embryonic lateral telencephalic neuroectoderm reveal that exogenous BMP proteins (BMP4 and BMP2) induce expression of Msx1 and inhibit Bf1 expression, a finding consistent with their specific expression patterns in vivo. Moreover, BMP proteins locally inhibit cell proliferation and increase apoptosis in the explants. These results provide evidence that BMPs function during regional morphogenesis of the dorsal telencephalon by regulating specific gene expression, cell proliferation and local cell death.

Animals↗

PubMed1996

The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.

One blastomere of the two-cell stage sea urchin embryo (Lytechinus variegatus) was labeled with an intracellular fluorescent lineage tracing stain to determine, from the lineage of that blastomere, the orientation of the first cleavage furrow with regard to the axes of bilateral symmetry in the gastrula and pluteus larva. Two methods were used to mark the blastomere: in the first, the lipophilic carbocyanine dye DiIC16 was microinjected directly into the blastomere after first cleavage was completed. In the second, caged (nonfluorescent) fluorescein-dextran was microinjected into the single-celled zygote and uncaged (made fluorescent) in one of the blastomeres at the two-cell stage using two-photon excitation microscopy (TPEM). This is the first use of TPEM for embryonic lineage tracing. In both methods the dye proved to be nontoxic and fluorescence was confined to lineally related cells. The results from both methods were similar and showed that the first cleavage furrow was variable in its orientation. Results were similar using animals obtained from different geographic locations. These results differ from those of McCain and McClay (Development, 1994, 120, 395-404), who reported that the median orientation was invariant in this species. The differences between the two studies are discussed. We conclude that first cleavage does not specify nor is it predictive of the bilateral axes in this species. The technique of TPEM is proffered as a powerful new tool that will enable the marking and tracing of embryonic cell lineages with less injury and more precision than current methods.

Animals↗

PubMed1996

Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets.

Studies of dispersed beta cells have been used to infer their behavior in the intact pancreatic islet. When dispersed, beta cells exhibit multiple metabolic glucose-response populations with different insulin secretion properties. This has led to a model for glucose-dependent insulin secretion from the islet based on a step-wise recruitment of individual beta cells. However, previously reported synchronous and uniform Ca2+ activity and electrical responses indicate that beta cell behavior within intact islets is more uniform. Therefore, uncertainty remains whether beta cell metabolic heterogeneity is functionally important in intact islets. We have used two-photon excitation microscopy to measure and compare the glucose-induced NAD(P)H autofluorescence response in dispersed beta cells and within intact islets. Over 90% of beta cells in intact islets responded to glucose with significantly elevated NAD(P)H levels, compared with less than 70% of dispersed beta cells. In addition, all responding beta cells within intact islets exhibited a sigmoidal glucose dose response behavior with inflection points of approximately 8 mm glucose. These results suggest that beta cell heterogeneity may be functionally less important in the intact islet than has been predicted from studies of dispersed beta cells and support the role of glucokinase as the rate-limiting enzyme in the beta cell glucose response.

Animals↗

PubMed1996

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1996

Ouabain increases aldosterone release from bovine adrenal glomerulosa cells: role of renin-angiotensin system.

To evaluate the potential physiological significance of ouabain or a ouabainlike substance, we investigated the effect of nanomolar concentrations of ouabain on aldosterone release by cultured bovine adrenal glomerulosa cells. Ouabain (10 nM) increased aldosterone release from 0.35 to 0.89 ng.mg-1.4 h-1 in the serum-containing medium. Losartan prevented this increase. When angiotensinogen was added to the nonserum medium, 10 nM ouabain enhanced the aldosterone release. Losartan again blocked the increase. These findings together with a stimulation of renin release by ouabain indicate that angiotensin II generated by the adrenal cell renin-angiotensin system in the presence of exogenous serum or exogenous angiotensinogen is necessary for the ouabain-induced stimulation of aldosterone release. Ouabain (10 nM) enhanced the intracellular calcium concentration increase elicited by 0.1 nM angiotensin II severalfold. Addition of 1 nM ouabain enhanced the aldosterone secretion resulting from the addition of 1 nM angiotensin II. Nanomolar levels of ouabain, therefore, interact with both locally formed and exogenous angiotensin II to stimulate aldosterone production. A suggested mechanism is that ouabain increases calcium stores in the endoplasmic reticulum, thereby increasing the agonist response.

Aldosterone↗

PubMed1995

Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy.

Three-dimensional maps of cellular metabolic oxidation/reduction states of rabbit cornea in situ were obtained by imaging the fluorescence of the naturally occurring reduced pyridine nucleotides (both reduced nicotinamide-adenine dinucleotide, NADH, and reduced nicotinamide-adenine dinucleotide phosphate, NADPH, denoted here as NAD(P)H). Autofluorescence images with submicrometre lateral resolution were obtained throughout the entire 400 microns thickness of the cornea. Two-photon excitation scanning laser microscopy with near-infrared excitation provided high fluorescence collection efficiency, reduced photodamage, and eliminated ultraviolet chromatic aberration, all of which have previously degraded the visualization of pyridine nucleotide fluorescence. Sharp autofluorescence images of the basal epithelium (40 microns within the cornea) show substantial subcellular detail, providing the ability to monitor autofluorescence intensity changes over time, which reflect changes in oxidative metabolism and cellular dynamics necessary for maintenance of the ocular surface. The autofluorescence was confirmed to be mostly of NAD(P)H origin by cyanide exposure, which increased the fluorescence from all cell types in the cornea by about a factor of two. Autofluorescence images of individual keratocytes in the stroma were observed only after cyanide treatment, while in the predominant extracellular collagen (> 90% of the stromal volume), fluorescence was not distinguished from the background. Observation of keratocyte metabolism demonstrates the sensitivity made available by two-photon microscopy for future redox fluorescence imaging of cellular metabolic states.

Animals↗

PubMed1995

Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits.

To determine the application limits of green fluorescent protein (GFP) as a reporter gene or protein tag, we expressed GFP by itself and with fusion protein partners, and used three different imaging methods to identify GFP fluorescence. In conventional epifluorescence photomicroscopy, GFP expressed in cells could be distinguished as a bright green signal over a yellow-green autofluorescence background. In quantitative fluorescence microscopy, however, the GFP signal is contaminated by cellular autofluorescence. Improved separation of GFP signal from HeLa cell autofluorescence was achieved by the combination of confocal scanning laser microscopy using 488-nm excitation, a rapid cut-on dichroic mirror and a narrow-bandpass emission filter. Two-photon excitation of GFP fluorescence at the equivalent of approximately 390 nm provided better absorption than did 488-nm excitation. This resulted in increased signal/background but also generated a different autofluorescence pattern and appeared to increase GFP photobleaching. Fluorescence spectra similar to those of GFP alone were observed when GFP was expressed as a fusion protein either with glutathione-S-transferase (GST) or with glucokinase. Furthermore, purified GST.GFP fusion protein displayed an extinction coefficient and quantum yield consistent with values previously reported for GFP alone. In HeLa cells, the cytoplasmic GFP concentration must be greater than approximately 1 microM to allow quantifiable discrimination over autofluorescence. However, lower expression levels may be detectable if GFP is targeted to discrete subcellular compartments, such as the plasma membrane, organelles or nucleus.

Base Sequence↗

PubMed1994

Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry.

With the development of sensitive and specific fluorescent indicators, modern laser scanning microscopies enable visualization and measurement of submicron, dynamic processes inside living cells and tissues. Here we describe the working principles of new, nonlinear laser microscopies based on two-photon molecular excitation. In these techniques, a pulsed laser produces peak photon densities high enough that when focused into an appropriate medium, excitation by photon energy combinations can occur. For example, two red photons interacting simultaneously with a fluorescent molecule can excite within it a UV electronic transition, one corresponding to twice the energy of each single photon. Because the amount of two-photon excitation depends on the square of the local illumination intensity, this process exhibits a unique localization to the diffraction-limited spot of the beam focus. Elsewhere along the beam, excitation of background and photodamage is virtually nonexistent. Focal point localization of two-photon excitation lends to all visualization, measurement, and photopharmacology studies an intrinsic, three-dimensional resolution. We describe some preliminary biological applications, specifically, imaging of vital DNA stains in developing cells and embryos, imaging of cellular metabolic activity from NADH autofluorescence, spatially resolved measurements of cytoplasmic calcium ion activity, and optically induced micropharmacology using caged bioeffector molecules.

Animals↗

PubMed1994

A confocal laser scanning microscope designed for indicators with ultraviolet excitation wavelengths.

In this paper we describe the modifications necessary to upgrade, at affordable cost, a commercially available confocal laser scanning microscope for use with ultraviolet (UV) excitation. The optical problems associated with these modifications are described in detail, and easy solutions to solve them are suggested. The optical resolution of the instrument was tested with fluorescent beads and was found to be close to diffraction limited. The light losses due to lateral chromatic aberration were assessed in a thick fluorescent specimen and were found to be comparable to those usually observed with visible light. For a more visual example of the resolution of this instrument, isolated ventricular heart muscle cells were loaded with the fluorescent Ca2+ indicator indo 1. This allowed us to visualize subcellular structural detail and to illustrate the optical sectioning capability of the UV confocal microscope when recording indo 1 emission. Dual-emission line scans were used to perform ratiometric time-resolved detection of Ca2+ transients in voltage-clamped heart muscle cells loaded with the salt form of indo 1. The system presented in this paper should significantly broaden the range of fluorescent indicators that can be used in confocal microscopy.

Animals↗

PubMed1989

Orientational exchange approach to fluorescence anisotropy decay.

Fluorescence depolarization is a powerful technique in resolving dynamics of molecular systems. Data obtained in fluorescence depolarization experiments are highly complex. Mathematical models for analyzing data from depolarization due to rotational motion have been largely based on the rotational diffusion equation. These results have been verified by Monte Carlo simulations. It has been implicitly stated that a 90 degrees jump model between predefined orientations such as presented by G. Weber (1971. J. Chem. Phys. 55:2399-2411) should, for the specific case of fluorescence depolarization, give the same answer as the diffusion equation. Since the highly symmetric cases considered by G. Weber gave the same result as the diffusion equation, it has been desirable to use this method in cases where depolarization arises from both discrete processes and rotational diffusion. We have derived, in a compartmental formalism, the general result for excitation and emission dipoles not necessarily coincident with any of the principal rotational axes of the fluorophore from this exchange model, and have found it to be different from that of the diffusion equation approach. We have also verified this difference with a Monte Carlo simulation of our exchange model. This derivation allows us to define the limits of validity of the 90 degrees exchanges to model rotational diffusion. Also, for systems where movements may be jumps between a few preferred orientations, the actual physical mechanism of depolarization may not be accurately represented by continuous diffusion. The compartmental formalism developed here can be used to easily combine rotational motions with discrete position jumps or other level kinetics. While the difference between the diffusion equation and random walk of finite step size derivations has been presented for observations of different order properties for the compartmental formalism, we discuss the possibility of finding this difference by using the ratio of relaxation rates from a single experiment. Also,the temperature dependence of the exchange rates is calculated in relation to the Kramer's theory.

Biopolymers↗