Biomedical subjects

Vytautas P Bindokas

Publications and source records attributed to Vytautas P Bindokas.

10 recordsLinked to original sources

PubMed2006

Flow cytometric analysis of phosphorylated histone H2AX following exposure to ionizing radiation in human microvascular endothelial cells.

We applied a flow cytometric method to quantify IR-induced histone H2AX phosphorylation at serine 139 (gammaH2AX) and compared those values to those obtained using a standard microscopy based foci counting method. After PFA fixation, methanol permeabilization was suitable for both FITC- or Alexa647-gammaH2AX. In contrast, Alexa647-gammaH2AX was not suitable for ethanol permeabilization. Antibody concentrations at 1-2 microg/ml yielded the highest gammaH2AX positive percentage for both antibodies. Without DAPI staining, gammaH2AX formation can be measured as a relative fold increase. Values determined by bivariant flow cytometric analysis and those obtained using microscopic foci formation exhibited a good quantitative correlation. Values obtained by both methods could vary according to the gating or threshold setting used. gammaH2AX positive cells increased as a function of radiation dose (2-16 Gy) followed by a dose-dependent decay. The free radical scavenger N-acetyl-L-cysteine (NAC), if administered at a concentration of 4 mM 30 min before IR, was effective in reducing IR-induced gammaH2AX formation in all phases of the cell cycle. We have developed a simplified and quantitative flow cytometry based method to measure IR-induced gammaH2AX in cells and demonstrated strong correlation to values obtained by a standard automated digital microscopic foci analysis along with NIH ImageJ custom macro software.

Cells, Cultured↗

PubMed2006

Visualizing calcium signaling in cells by digitized wide-field and confocal fluorescent microscopy.

Calcium (Ca2+) is a fundamentally important component of cellular signal transduction. Dynamic changes in the concentration of Ca2+ ([Ca2+]) in the cytoplasm and within organelles are tightly controlled and regulate a diverse array of biological activities, including fertilization, cell division, gene expression, cellular metabolism, protein biosynthesis, secretion, muscle contraction, intercellular communication, and cell death. Measurement of intracellular [Ca2+] is essential to understanding the role of Ca2+ and for defining the underlying regulatory mechanisms in any cellular process. A broad range of synthetic and biosynthetic fluorescent Ca2+ sensors are available that enable the visualization and quantification of subcellular spatio-temporal [Ca2+] gradients. This chapter describes the application of wide-field digitized video fluorescence microfluorometry and confocal microscopy to quantitatively image Ca2+ in cells with high temporal and spatial resolution.

Animals↗

PubMed2005

Imaging pancreatic beta-cells in the intact pancreas.

We have developed a method to visualize fluorescent protein-labeled beta-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the beta-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, beta-cell clusters, and single beta-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary beta-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red beta-cells. The imaging technique that we have developed, coupled with the transgenic mice in which beta-cells and beta-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states.

Animals↗

PubMed2005

FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets.

Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (V(m)) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from V(m) -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in V(m). When V(m) was measured simultaneously with intracellular Ca2+, all active cells showed tight coupling of V(m) to islet cell Ca2+ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans.

Animals↗

PubMed2005

Inositol (1,4,5)-trisphosphate dynamics and intracellular calcium oscillations in pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with transients of intracellular calcium concentration ([Ca2+]i) in the pancreatic beta-cell. We tested the hypothesis that inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] [Ca2+]i release is incorporated in glucose-induced [Ca2+]i oscillations in mouse islets and MIN6 cells. We found that depletion of intracellular Ca2+ stores with thapsigargin increased the oscillation frequency by twofold and inhibited the slow recovery phase of [Ca2+]i oscillations. We employed a pleckstrin homology domain-containing fluorescent biosensor, phospholipase C partial differential pleckstrin homology domain-enhanced green fluorescent protein, to visualize Ins(1,4,5)P3 dynamics in insulin-secreting MIN6 cells and mouse islets in real time using a video-rate confocal system. In both types of cells, stimulation with carbamoylcholine (CCh) and depolarization with KCl results in an increase in Ins(1,4,5)P3 accumulation in the cytoplasm. When stimulated with glucose, the Ins(1,4,5)P3 concentration in the cytoplasm oscillates in parallel with oscillations of [Ca2+]i. Maximal accumulation of Ins(1,4,5)P3 in these oscillations coincides with the peak of [Ca2+]i and tracks changes in frequencies induced by the voltage-gated K+ channel blockade. We show that Ins(1,4,5)P3 release in insulin-secreting cells can be stimulated by depolarization-induced Ca2+ flux. We conclude that Ins(1,4,5)P3 concentration oscillates in parallel with [Ca2+]i in response to glucose stimulation, but it is not the driving force for [Ca2+]i oscillations.

Animals↗

PubMed2004

Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion.

Confocal imaging of GFP-tagged secretory granules combined with the use of impermeant extracellular dyes permits direct observation of insulin packaged in secretory granules, trafficking of these granules to the plasma membrane, exocytotic fusion of granules with the plasma membrane, and eventually the retrieval of membranes by endocytosis. Most such studies have been done in tumor cell lines, using either confocal methods or total internal reflectance microscopy. Here we compared these methods by using GFP-syncollin or PC3-GFP plus rhodamine dextrans to study insulin granule dynamics in insulinoma cells, normal mouse islets, and primary pancreatic beta cells. We found that most apparently docked granules did not fuse with the plasma membrane after stimulation. Granules that did fuse typically fused completely, but a few dextran-filled granules lingered at the membrane. Direct recycling of granules occurred only rarely. Similar results were obtained with both confocal and total internal reflection microscopy, although each technique had advantages for particular aspects of the granule life cycle. We conclude that insulin exocytosis involves a prolonged interaction of secretory granules with the plasma membrane, and that the majority of exocytotic events occur by full, not partial, fusion.

Animals↗

PubMed2003

Visualizing superoxide production in normal and diabetic rat islets of Langerhans.

Oxygen free radicals have been implicated in beta-cell dysfunction and apoptosis associated with type 1 and type 2 diabetes mellitus. The roles of free radicals in diabetes have thus far been defined indirectly by monitoring oxidative tissue damage and the effects of antioxidants, free radical scavengers, and overexpression of superoxide dismutase. We employed the superoxide-mediated oxidation of hydroethidine to ethidium to dynamically and directly assess the relative rates of mitochondrial superoxide anion generation in isolated islets in response to glucose stimulation. Superoxide content of isolated islets increased in response to glucose stimulation. We next compared the oxyradical levels in Zucker lean control and Zucker diabetic fatty rat islets by digital imaging microfluorometry. The superoxide content of Zucker diabetic fatty islets was significantly higher than Zucker lean control islets under resting conditions, relatively insensitive to elevated glucose concentrations, and correlated temporally with a decrease in glucose-induced hyperpolarization of the mitochondrial membrane. Importantly, superoxide levels were elevated in islets from young, pre-diabetic Zucker diabetic fatty animals. Overproduction of superoxide was associated with perturbed mitochondrial morphology and may contribute to abnormal glucose signaling found in the Zucker diabetic fatty model of type 2 diabetes mellitus.

Animals↗

PubMed2003

A 48-hour exposure of pancreatic islets to calpain inhibitors impairs mitochondrial fuel metabolism and the exocytosis of insulin.

Genetic variation in the gene for a cytosolic cysteine protease, calpain-10, increases the susceptibility to type 2 diabetes apparently by altering levels of gene expression. In view of the importance of altered beta-cell function in the pathophysiology of type 2 diabetes, the present study was undertaken to define the effects on insulin secretion of exposing pancreatic islets to calpain inhibitors for 48 hours. Exposure of mouse islets to calpain inhibitors (ALLN, ALLM, E-64-d, MDL 18270, and PD147631) of different structure and mechanism of action for 48 hours reversibly suppresses glucose-induced insulin secretion by 40% to 80%. Exposure of islets to inhibitors of other proteases, ie, cathepsin B and proteasome, did not affect insulin secretion. The 48-hour incubation with calpain inhibitors also attenuates insulin secretory responses to the mitochondrial fuel alpha-ketoisocaproate (KIC). The same incubation also suppresses glucose metabolism and intracellular calcium ([Ca(2+)](i)) responses to glucose or KIC in islets. In summary, long-term inhibition of islet calpain activity attenuates insulin secretion possibly by limiting the rate of glucose metabolism. A reduction of calpain activity in islet could contribute to the development of beta-cell failure in type 2 diabetes thereby providing a link between genetic susceptibility to diabetes and the pathophysiologic manifestations of the disease.

Animals↗

PubMed2002

Bcl-X(L) affects Ca(2+) homeostasis by altering expression of inositol 1,4,5-trisphosphate receptors.

An oligonucleotide-based microarray analysis of 9,500 genes and expressed sequence tags (ESTs) demonstrated that the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R) was significantly down-regulated in Bcl-X(L)-expressing as compared with control cells. This result was confirmed at the mRNA and protein levels by Northern and Western blot analyses of two independent hematopoietic cell lines and murine primary T cells. Bcl-X(L) expression resulted in a dose-dependent decrease in IP(3)R protein. IP(3)R expression is regulated as part of a mitochondrion-to-nucleus stress-responsive pathway. The uncoupling of mitochondrial oxidative phosphorylation resulted in induction of binding of the transcription factor NFATc2 to the IP(3)R promoter and transcriptional activation of IP(3)R. Expression of Bcl-X(L) led to a decreased induction of both NFATc2 DNA binding to the IP(3)R promoter and IP(3)R expression in response to the inhibition of mitochondrial oxidative phosphorylation. The Bcl-X(L)-dependent decrease in IP(3)R expression also correlated with a reduced T cell antigen receptor ligation-induced Ca(2+) flux in Bcl-X(L) transgenic murine T cells, and microsomal vesicles prepared from Bcl-X(L)-overexpressing cells exhibited lower IP(3)-mediated Ca(2+) release capacity. Furthermore, reintroducing IP(3)R into Bcl-X(L)-transfected cells partially reversed Bcl-X(L)-dependent anti-apoptotic activity. These results suggest that even under non-apoptotic conditions, expression of Bcl-2-family proteins influences a signaling network that links changes in mitochondrial metabolism to alterations in nuclear gene expression.

Animals↗

PubMed2002

Polyglutamine protein aggregates are dynamic.

Protein aggregation and the formation of inclusion bodies are hallmarks of the cytopathology of neurodegenerative diseases, including Huntington's disease, Amyotropic lateral sclerosis, Parkinson's disease and Alzheimer's disease. The cellular toxicity associated with protein aggregates has been suggested to result from the sequestration of essential proteins that are involved in key cellular events, such as transcription, maintenance of cell shape and motility, protein folding and protein degradation. Here, we use fluorescence imaging of living cells to show that polyglutamine protein aggregates are dynamic structures in which glutamine-rich proteins are tightly associated, but which exhibit distinct biophysical interactions. In contrast, the interaction between wild-type, but not mutant, Hsp70 exhibits rapid kinetics of association and dissociation similar to interactions between Hsp70 and thermally unfolded substrates. These studies provide new insights into the composite organization and formation of protein aggregates and show that molecular chaperones are not sequestered into aggregates, but are instead transiently associated.

Animals↗