Biomedical subjects

James D Lechleiter

Publications and source records attributed to James D Lechleiter.

9 recordsLinked to original sources

PubMed2006

Multiphoton laser scanning microscopy as a tool for Xenopus oocyte research.

Multiphoton laser scanning microscopy (MPLSM) has become an increasingly invaluable tool in fluorescent optical imaging. There are several distinct advantages to implementing MPLSM as a Xenopus oocyte research tool. MPLSM increases signal-to-noise ratio and therefore increases image quality because there is no out-of-focus fluorescence as would be created in conventional or confocal microscopy. All the light that is generated can be collected and used to generate an image because point detection of descanned fluorescence is not required. This is particularly useful when imaging deep into tissue sections, as is necessary for Xenopus oocytes, which are notoriously large (approximately 1-mm diameter). Because multiphoton lasers use pulsed energy in the infrared wavelengths, the energy can also travel further into tissues with much less light scattering. Because there is no out-of-focus excitation, phototoxicity, photodamage, and photobleaching are significantly reduced, which is particularly important for long-term experiments that require the same region to be scanned repeatedly. Finally, multiple fluorophores can be simultaneously excited because of the broader absorption spectra of multiphoton dyes. In this chapter, we describe the advantages and disadvantages of using MPLSM to image Xenopus oocytes as compared to conventional and confocal microscopy. The practical application of imaging oocytes is demonstrated with specific examples.

Animals↗

PubMed2005

Ca2+ signaling, mitochondria and sensitivity to oxidative stress in aging astrocytes.

Age-related changes in astrocytes that could potentially affect neuroprotection have been largely unexplored. To test whether astrocyte function was diminished during the aging process, we examined cell growth, Ca2+ signaling, mitochondrial membrane potential (DeltaPsi) and neuroprotection of NGF-differentiated PC12 cells. We observed that cell growth was significantly slower for astrocytes cultured from old (26-29 months) mice as compared to young (4-6 months) mice. DeltaPsis in old astrocytes were also more depolarized (lower) than in young astrocytes and old astrocytes showed greater sensitivity to the oxidant tert-butyl hydrogen peroxide (t-BuOOH). ATP-induced Ca2+ responses in old astrocytes were consistently larger in amplitude and more frequently oscillatory than in young astrocytes, which may be attributable to lower mitochondrial Ca2+ sequestration. Finally, NGF-differentiated PC12 cells that were co-cultured with old astrocytes were significantly more sensitive to t-BuOOH treatment than co-cultures of NGF-differentiated PC12 cells with young astrocytes. Together, these data demonstrate that astrocyte physiology is significantly altered during the aging process and that the astrocyte's ability to protect neurons is compromised.

Aging↗

PubMed2004

Nontranscriptional modulation of intracellular Ca2+ signaling by ligand stimulated thyroid hormone receptor.

Thyroid hormone 3,5,3'-tri-iodothyronine (T3) binds and activates thyroid hormone receptors (TRs). Here, we present evidence for a nontranscriptional regulation of Ca2+ signaling by T3-bound TRs. Treatment of Xenopus thyroid hormone receptor beta subtype A1 (xTRbetaA1) expressing oocytes with T3 for 10 min increased inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ wave periodicity. Coexpression of TRbetaA1 with retinoid X receptor did not enhance regulation. Deletion of the DNA binding domain and the nuclear localization signal of the TRbetaA1 eliminated transcriptional activity but did not affect the ability to regulate Ca2+ signaling. T3-bound TRbetaA1 regulation of Ca2+ signaling could be inhibited by ruthenium red treatment, suggesting that mitochondrial Ca2+ uptake was required for the mechanism of action. Both xTRbetaA1 and the homologous shortened form of rat TRalpha1 (rTRalphaDeltaF1) localized to the mitochondria and increased O2 consumption, whereas the full-length rat TRalpha1 did neither. Furthermore, only T3-bound xTRbetaA1 and rTRalphaDeltaF1 affected Ca2+ wave activity. We conclude that T3-bound mitochondrial targeted TRs acutely modulate IP3-mediated Ca2+ signaling by increasing mitochondrial metabolism independently of transcriptional activity.

Animals↗

PubMed2003

Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism.

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA overflow into the synaptic cleft. Facilitated exchange diffusion is the classical model used to describe AMPH-induced DA efflux. This model hypothesizes that AMPH-induced DA efflux is mediated by DAT and results from the transport of AMPH into the cell followed by a counter movement of DA out to the extracellular compartment. To further characterize the action of AMPH, we used the patch clamp technique in the whole-cell configuration combined with amperometry on human embryonic kidney HEK-293 cells stably transfected with the human DAT (DAT cells). In DAT cells, AMPH-induced DAT-mediated currents were blocked by cocaine. We demonstrate that DA efflux mediated by DAT is voltage-dependent, electrogenic, and dependent on intracellular Na(+) concentration in the recording electrode. Intracellular Na(+) fluorescence, as measured by confocal microscopy using a Na(+)-sensitive dye, was enhanced by AMPH application. Furthermore, the ability of AMPH to induce DA efflux was regulated by intracellular Na(+) concentration and correlated with the size of the DAT-mediated, AMPH-induced ion flux across the plasma membrane. In the absence of intracellular Na(+) but the presence of high intracellular Cl(-), AMPH-induced inward currents elicited DA efflux proportionally to their dimension and duration. Thus, we propose that AMPH-induced DA efflux depends on two correlated transporter processes. First, AMPH binds to the DAT and is transported, thereby causing an inward current. Second, because of this AMPH-induced inward current, Na(+) becomes more available intracellularly to the DAT, thereby enhancing DAT-mediated reverse transport of DA.

Amphetamine↗

PubMed2003

Modeling the dependence of the period of intracellular Ca2+ waves on SERCA expression.

Contrary to intuitive expectations, overexpression of sarco-endoplasmic reticulum (ER) Ca(2+) ATPases (SERCAs) in Xenopus oocytes leads to a decrease in the period and an increase in the amplitude of intracellular Ca(2+) waves. Here we examine these experimental findings by modeling Ca(2+) release using a modified Othmer-Tang-model. An increase in the period and a reduction in the amplitude of Ca(2+) wave activity are obtained when increases in SERCA density are simulated while keeping all other parameters of the model constant. However, Ca(2+) wave period can be reduced and the wave amplitude and velocity can be significantly increased when an increase in the luminal ER Ca(2+) concentration due to SERCA overexpression is incorporated into the model. Increased luminal Ca(2+) occurs because increased SERCA activity lowers cytosolic Ca(2+), which is partially replenished by Ca(2+) influx across the plasma membrane. These simulations are supported by experimental data demonstrating higher luminal Ca(2+) levels, decreased periods, increased amplitude, and increased velocity of Ca(2+) waves in response to increased SERCA density.

Animals↗

PubMed2003

Role of the promyelocytic leukemia body in the dynamic interaction between the androgen receptor and steroid receptor coactivator-1 in living cells.

The dynamic interaction between the androgen receptor (AR) and steroid receptor coactivator-1 (SRC-1) was explored in living cells expressing chimeric forms of the receptor and the coactivator containing two spectral variants of jellyfish fluorescent protein. Laser scanning confocal imaging of transfected cells expressing fluorescently labeled SRC-1 revealed that in an unsynchronized cell population, the coactivator is distributed in approximately 40% cells as nuclear bodies of 0.2-1.0 microm in diameter. Immunostaining of cyan fluorescent protein-labeled SRC-1 (CFP-SRC1)-expressing cells with antibody to promyelocytic leukemia (PML) protein showed significant overlap of the CFP fluorescence with the antibody stain. Cotransfection of cells with a plasmid expressing the CFP conjugate of Sp100 (another marker protein for the PML nuclear body) also showed colocalization of the yellow fluorescent protein (YFP)-SRC1 containing nuclear foci with the PML bodies in living cells. Analysis of the three-dimensional structure revealed that the PML bodies are round to elliptical in shape with multiple satellite bodies on their surface. Some of these satellite bodies contain the SRC-1. Activation and nuclear import of CFP-AR by the agonistic ligand 5alpha-dihydrotestosterone, but not by the antagonist casodex, transferred YFP-SRC1 from the PML bodies to an interlacing filamentous structure. In a single living cell, agonist-activated AR caused a time-dependent movement of YFP-SRC1 from the PML bodies to this filamentous structure. Additionally, coexpression of a constitutively active mutant of AR (AR-deltaligand binding domain) also displaced YFP-SRC1 from the PML bodies to this intranuclear filamentous structure. The fluorescence recovery after photobleaching approach was used to examine changes in the kinetics of movement of YFP-SRC1 during its mobilization from the PML bodies to the intranuclear filamentous structure by the agonist-activated AR. Results of the relative half-times (t(1/2)) of replacement of YFP-SRC1 within the photobleached region of a single PML body from its surrounding nuclear space supported the conclusion that SRC-1 is actively transported from the PML bodies to the intranuclear filamentous structure by the ligand-activated AR. This observation also suggests an interaction between AR and SRC-1 before its binding to the target gene. The PML bodies have been implicated as a cross-road for multiple regulatory pathways that control cell proliferation, cellular senescence, and apoptosis. Our present results along with other recent reports expand the role of this subnuclear structure to include the regulation of steroid hormone action.

Active Transport, Cell Nucleus↗

PubMed2002

Mitochondrial targeted cyclophilin D protects cells from cell death by peptidyl prolyl isomerization.

Cyclophilin D (CyPD) is thought to sensitize opening of the mitochondrial permeability transition pore (mPTP) based on the findings that cyclosporin A (CsA), a pseudo-CyPD substrate, hyperpolarizes the mitochondrial membrane potential (DeltaPsi) and inhibits apoptosis. We provide evidence that contrasts with this model. Using live cell imaging and two photon microscopy, we report that overexpression of CyPD desensitizes HEK293 and rat glioma C6 cells to apoptotic stimuli. By site-directed mutagenesis of CyPD that compromises peptidyl-prolyl cis-trans isomerase (PPIase) activity, we demonstrate that the mechanism involved in this protective effect requires PPIase activity. Furthermore, we show that, under resting conditions, DeltaPsi is hyperpolarized in CyPD wild type-overexpressing cells but not in cells overexpressing mutant forms of CyPD that lack PPIase activity. Finally, in glutathione S-transferase (GST) pull-down assays, we demonstrate that CyPD binding to the adenine nucleotide translocator (ANT), which is considered to be the core component of the mPTP, is not affected by the loss of PPIase activity. Collectively, our data suggest that CyPD should be viewed as a cell survival-signaling molecule and indicate a protective role of CyPD against apoptosis that is mediated by one or more targets other than the ANT.

Animals↗

PubMed2002

Multi-photon laser scanning microscopy using an acoustic optical deflector.

Multi-photon laser scanning microscopes have many advantages over single-photon systems. However, the speed and flexibility of currently available multi-photon microscopes are limited by the use of mechanical mirrors to steer pulsed radiation for fluorophore excitation. Here, we describe the multi-photon adaptation of a confocal microscope that uses an acoustic optical deflector (AOD) for beam steering. AODs are capable of very rapid scanning and, in addition, offer the flexibility of zooming, panning, and being adjustable for slow image acquisition. Because of the highly dispersive nature of AODs, pulsed radiation must be temporally compressed by introducing negative dispersion into the beam path. More critically, pulsed radiation must also be spatially compressed by introducing lateral dispersion into the beam path. This was accomplished by using two prisms in the external beam path and by introducing a third prism element subsequent to the AOD. The end result is an AOD-based multi-photon microscope that is capable of rapid imaging of physiological events as well as slow detection of weakly fluorescent biological samples.

Animals↗

PubMed2002

PI 3-kinase regulation of dopamine uptake.

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3-kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [(3) H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3-kinase, increased [(3)H]DA uptake and blocked the amphetamine-induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3-kinase. The LY294002-induced reduction in [(3)H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin-dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.

Amphetamine↗