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Before insulin can stimulate myocytes to take up glucose, it must first move from the circulation to the interstitial space. The continuous endothelium of skeletal muscle (SkM) capillaries restricts insulin's access to myocytes. The mechanism by which insulin crosses this continuous endothelium is critical to understand insulin action and insulin resistance; however, methodological obstacles have limited understanding of endothelial insulin transport in vivo. Here, we present an intravital microscopy technique to measure the rate of insulin efflux across the endothelium of SkM capillaries. This method involves development of a fully bioactive, fluorescent insulin probe, a gastrocnemius preparation for intravital microscopy, an automated vascular segmentation algorithm, and the use of mathematical models to estimate endothelial transport parameters. We combined direct visualization of insulin efflux from SkM capillaries with modeling of insulin efflux kinetics to identify fluid-phase transport as the major mode of transendothelial insulin efflux in mice. Model-independent experiments demonstrating that insulin movement is neither saturable nor affected by insulin receptor antagonism supported this result. Our finding that insulin enters the SkM interstitium by fluid-phase transport may have implications in the pathophysiology of SkM insulin resistance as well as in the treatment of diabetes with various insulin analogs.

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid β1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Increasing evidence indicates that proteotoxicity plays a pathophysiologic role in experimental and human cardiomyopathy. In organ-specific amyloidoses, soluble protein oligomers are the primary cytotoxic species in the process of protein aggregation. While isolated atrial amyloidosis can develop with aging, the presence of preamyloid oligomers (PAOs) in atrial tissue has not been previously investigated., Methods and Results: Atrial samples were collected during elective cardiac surgery in patients without a history of atrial arrhythmias, congestive heart failure, cardiomyopathy, or amyloidosis. Immunohistochemistry was performed for PAOs using a conformation-specific antibody, as well as for candidate proteins identified previously in isolated atrial amyloidosis. Using a myocardium-specific marker, the fraction of myocardium colocalizing with PAOs (PAO burden) was quantified (green/red ratio). Atrial samples were obtained from 92 patients, with a mean age of 61.7±13.8 years. Most patients (62%) were male, 23% had diabetes, 72% had hypertension, and 42% had coronary artery disease. A majority (n=62) underwent aortic valve replacement, with fewer undergoing coronary artery bypass grafting (n=34) or mitral valve replacement/repair (n=24). Immunostaining detected intracellular PAOs in a majority of atrial samples, with a heterogeneous distribution throughout the myocardium. Mean green/red ratio value for the samples was 0.11±0.1 (range 0.03 to 0.77), with a value ≥0.05 in 74 patients. Atrial natriuretic peptide colocalized with PAOs in myocardium, whereas transthyretin was located in the interstitium. Adjusting for multiple covariates, PAO burden was independently associated with the presence of hypertension., Conclusion: PAOs are frequently detected in human atrium, where their presence is associated with clinical hypertension., (© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Abnormalities in atrial myocardium increase the likelihood of arrhythmias, including atrial fibrillation (AF). The deposition of misfolded protein, or amyloidosis, plays an important role in the pathophysiology of many diseases, including human cardiomyopathies. We have shown that genes implicated in amyloidosis are activated in a cellular model of AF, with the development of preamyloid oligomers (PAOs). PAOs are intermediates in the formation of amyloid fibrils, and they are now recognized to be the cytotoxic species during amyloidosis. To investigate the presence of PAOs in human atrium, we developed a microscopic imaging-based protocol to enable robust and reproducible quantitative analysis of PAO burden in atrial samples harvested at the time of elective cardiac surgery. Using PAO- and myocardial-specific antibodies, we found that PAO distribution was typically heterogeneous within a myocardial sample. Rigorous imaging and analysis protocols were developed to quantify the relative area of myocardium containing PAOs, termed the Green/Red ratio (G/R), for a given sample. Using these methods, reproducible G/R values were obtained when different sections of a sample were independently processed, imaged, and analyzed by different investigators. This robust technique will enable studies to investigate the role of this novel structural abnormality in the pathophysiology of and arrhythmia generation in human atrial tissue., (© The Author(s) 2014.)

Environmental exposure to manganese (Mn) leads to a neurodegenerative disease that has shared clinical characteristics with Parkinson's disease (PD). Mn-induced neurotoxicity is time- and dose-dependent, due in part to oxidative stress. We ascertained the molecular targets involved in Mn-induced neurodegeneration using astrocyte culture as: (1) Astrocytes are vital for information processing within the brain, (2) their redox potential is essential in mitigating reactive oxygen species (ROS) levels, and (3) they are targeted early in the course of Mn toxicity. We first tested protein levels of Mn superoxide dismutase -2 (SOD-2) and glutathione peroxidase (GPx-1) as surrogates of astrocytic oxidative stress response. We assessed levels of the forkhead winged-helix transcription factor O (FoxO) in response to Mn exposure. FoxO is highly regulated by the insulin-signaling pathway. FoxO mediates cellular responses to toxic stress and modulates adaptive responses. We hypothesized that FoxO is fundamental in mediating oxidative stress response upon Mn treatment, and may be a biomarker of Mn-induced neurodegeneration. Our results indicate that 100 or 500 µM of MnCl2 led to increased levels of FoxO (dephosphorylated and phosphorylated) compared with control cells (P<0.01). p-FoxO disappeared from the cytosol upon Mn exposure. Pre-treatment of cultured cells with (R)-(-)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO. At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged. FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor). We conclude that FoxO phosphorylation after Mn exposure occurs in parallel with, and independent of the insulin-signaling pathway. FoxO levels and its translocation into the nucleus are part of early events compensating for Mn-induced neurotoxicity and may serve as valuable targets for neuroprotection in the setting of Mn-induced neurodegeneration.

Autoreactive B lymphocytes that are not culled by central tolerance in the bone marrow frequently enter the peripheral repertoire in a state of functional impairment, termed anergy. These cells are recognized as a liability for autoimmunity, but their contribution to disease is not well understood. Insulin-specific 125Tg B cells support T cell-mediated type 1 diabetes in NOD mice, despite being anergic to B cell mitogens and T cell-dependent immunization. Using this model, the potential of anergic, autoreactive B cells to present Ag and activate T cells was investigated. The data show that 1) insulin is captured and rapidly internalized by 125Tg BCRs, 2) these Ag-exposed B cells are competent to activate both experienced and naive CD4(+) T cells, 3) anergic 125Tg B cells are more efficient than naive B cells at activating T cells when Ag is limiting, and 4) 125Tg B cells are competent to generate low-affinity insulin B chain epitopes necessary for activation of diabetogenic anti-insulin BDC12-4.1 T cells, indicating the pathological relevance of anergic B cells in type 1 diabetes. Thus, phenotypically tolerant B cells that are retained in the repertoire may promote autoimmunity by driving activation and expansion of autoaggressive T cells via Ag presentation.

Na(+) current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na(+) channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na(+) currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na(+) channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca(2+)-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser(1503) or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na(+) channels in living cells.

Rationale: Voltage-gated sodium channels initiate action potentials in excitable tissues. Mice in which Scn5A (the predominant sodium channel gene in heart) has been knocked out die early in development with cardiac malformations by mechanisms which have yet to be determined., Objective: Here we addressed this question by investigating the role of cardiac sodium channels in zebrafish heart development., Methods and Results: Transcripts of the functionally-conserved Scn5a homologs scn5Laa and scn5Lab were detected in the gastrulating zebrafish embryo and subsequently in the embryonic myocardium. Antisense knockdown of either channel resulted in marked cardiac chamber dysmorphogenesis and perturbed looping. These abnormalities were associated with decreased expression of the myocardial precursor genes nkx2.5, gata4, and hand2 in anterior lateral mesoderm and significant deficits in the production of cardiomyocyte progenitors. These early defects did not appear to result from altered membrane electrophysiology, as prolonged pharmacological blockade of sodium current failed to phenocopy channel knockdown. Moreover, embryos grown in calcium channel blocker-containing medium had hearts that did not beat but developed normally., Conclusions: These findings identify a novel and possibly nonelectrogenic role for cardiac sodium channels in heart development.

The pancreatic islets of Langerhans are highly vascularized micro-organs that play a key role in the regulation of blood glucose homeostasis. The specific arrangement of endocrine cell types in islets suggests a coupling between morphology and function within the islet. Here, we established a line-scanning confocal microscopy approach to examine the relationship between blood flow and islet cell type arrangement by real-time in vivo imaging of intra-islet blood flow in mice. These data were used to reconstruct the in vivo 3D architecture of the islet and time-resolved blood flow patterns throughout the islet vascular bed. The results revealed 2 predominant blood flow patterns in mouse islets: inner-to-outer, in which blood perfuses the core of beta cells before the islet perimeter of non-beta cells, and top-to-bottom, in which blood perfuses the islet from one side to the other regardless of cell type. Our approach included both millisecond temporal resolution and submicron spatial resolution, allowing for real-time imaging of islet blood flow within the living mouse, which has not to our knowledge been attainable by other methods.

Objective: Na(+) current derived from expression of the principal cardiac Na(+) channel, Na(v)1.5, is increased by activation of protein kinase A (PKA). This effect is blocked by inhibitors of cell membrane recycling, or removal of a cytoplasmic endoplasmic reticulum (ER) retention motif, suggesting that PKA stimulation increases trafficking of cardiac Na(+) channels to the plasma membrane., Methods: To test this hypothesis, green fluorescent protein (GFP) was fused to Na(v)1.5 (Na(v)1.5-GFP), and the effects of PKA activation were investigated in intact, living cells that stably expressed the fusion protein. Using confocal microscopy, the spatial relationship of GFP-tagged channels relative to the plasma membrane was quantitated using a measurement that could control for variables present during live-cell imaging, and permit an unbiased analysis for all cells in a given field., Results: In the absence of kinase stimulation, intracellular fluorescence representing Na(v)1.5-GFP channels was greatest in the perinuclear area, with additional concentration of channels beneath the cell surface. Activation of PKA promoted trafficking of Na(+) channels from both regions to the plasma membrane. Experimental results using a chemiluminescence-based assay further confirmed that PKA stimulation increased expression of Na(v)1.5 channels at the cell membrane., Conclusions: Our results provide direct evidence for PKA-mediated trafficking of cardiac Na(+) channels into the plasma membrane in living, mammalian cells, and they support the existence of multiple intracellular storage pools of channel protein that can be mobilized following a physiologic stimulus.

ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine (A-to-I). ADAR2 contains two tandem double-stranded RNA-binding motifs (dsRBMs) that are not only important for efficient editing of RNA substrates but also necessary for localizing ADAR2 to nucleoli. The sequence and structural similarity of these motifs have raised questions regarding the role(s) that each dsRBM plays in ADAR2 function. Here, we demonstrate that the dsRBMs of ADAR2 differ in both their ability to modulate subnuclear localization as well as to promote site-selective A-to-I conversion. Surprisingly, dsRBM1 contributes to editing activity in a substrate-dependent manner, indicating that dsRBMs recognize distinct structural determinants in each RNA substrate. Although dsRBM2 is essential for the editing of all substrates examined, a point mutation in this motif affects editing for only a subset of RNAs, suggesting that dsRBM2 uses unique sets of amino acid(s) for functional interactions with different RNA targets. The dsRBMs of ADAR2 are interchangeable for subnuclear targeting, yet such motif alterations do not support site-selective editing, indicating that the unique binding preferences of each dsRBM differentially contribute to their pleiotropic function.

By using current biological understanding, a conceptually simple, but mathematically complex, model is proposed for the dynamics of the gene circuit responsible for regulating nitrogen catabolite repression (NCR) in yeast. A variety of mathematical "structure" theorems are described that allow one to determine the asymptotic dynamics of complicated systems under very weak hypotheses. It is shown that these theorems apply to several subcircuits of the full NCR circuit, most importantly to the URE2-GLN3 subcircuit that is independent of the other constituents but governs the switching behavior of the full NCR circuit under changes in nitrogen source. Under hypotheses that are fully consistent with biological data, it is proven that the dynamics of this subcircuit is simple periodic behavior in synchrony with the cell cycle. Although the current mathematical structure theorems do not apply to the full NCR circuit, extensive simulations suggest that the dynamics is constrained in much the same way as that of the URE2-GLN3 subcircuit. This finding leads to the proposal that mathematicians study genetic circuits to find new geometries for which structure theorems may exist.

Human small ubiquitin-like modifier (sumo) proteins include sumo-1 and the less studied, nearly identical sumo-2 and sumo-3 proteins. Whereas the structurally related ubiquitin molecule targets proteins for degradation, sumo provides a distinct, yet poorly understood regulatory signal. Protein sumoylation is sensitive to diverse cellular stresses, yet the targets of sumoylation in stress are unknown. We studied protein sumoylation in HEK293 cells exposed to hydrogen peroxide, alkylating agents, and the lipid oxidation-derived electrophile 4-hydroxynonenal, which is an ubiquitous product of lipid oxidation associated with oxidative stress. Confocal immunofluorescence microscopy indicated that in unstressed cells sumo-1 targeted nuclear proteins, whereas sumo-2/3 targeted proteins in both nuclei and cytoplasm. Western blot analyses revealed changes in sumo-1 and sumo-2/3 targeting patterns with stress. We used immunoaffinity chromatography to harvest sumo-associated proteins from HA-sumo-1- and HA-sumo-3-expressing HEK293 cells both before and after treatment with 4-hydroxynonenal. Multidimensional liquid chromatography-tandem mass spectrometry analyses identified 54 HA-sumo-1-associated proteins and 38 HA-sumo-3-associated proteins. Major protein targets included RNA binding and processing proteins, transcription factors, metabolic enzymes, and cytoskeletal regulators. Treatment with 4-hydroxynonenal caused a near-complete redistribution of sumo-1 and sumo-3 to different protein targets, which included chaperones, antioxidant, and DNA damage signaling proteins. A 10-15% overlap of sumo-1 and sumo-3 targets before and after stress suggests that sumo proteins target distinct protein groups. The results suggest that reactive electrophiles not only directly modify proteins but also lead to indirect changes in endogenous protein modifications that regulate protein functions.

Background: Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies., Results: A gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis. Four of the 111 genes disrupted in clones selected for resistance to infection by reovirus type 1 involved the insulin growth factor-2 (IGF-II) pathway, including: the mannose-6-phosphate/IGF2 receptor (Igf2r), a protease associated with insulin growth factor binding protein 5 (Prss11), and the CTCF transcriptional regulator (Ctcf). The disruption of Ctcf, which encodes a repressor of Igf2, was associated with enhanced Igf2 gene expression. Plasmids expressing either the IGF-II pro-hormone or IGF-II without the carboxy terminal extension (E)-peptide sequence independently conferred high levels of cellular resistance to reovirus infection. Forced IGF-II expression results in a block in virus disassembly. In addition, Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo., Conclusion: These results indicate that IGF-II, and by inference other components of the IGF-II signalling pathway, can confer resistance to lytic reovirus infection. This report represents the first use of gene entrapment to identify host factors affecting virus infection. Concomitant transformation observed in some virus resistant cells illustrates a potential mechanism of carcinogenesis associated with chronic virus infection.

The present study describes the in vivo detection and imaging of tumour-associated MMP-7 (matrix metalloproteinase-7 or matrilysin) activity using a novel polymer-based fluorogenic substrate PB-M7VIS, which serves as a selective 'proteolytic beacon' (PB) for this metalloproteinase. PB-M7VIS is built on a PAMAM (polyamido amino) dendrimer core of 14.2 kDa, covalently coupled with an Fl (fluorescein)-labelled peptide Fl(AHX)RPLALWRS(AHX)C (where AHX stands for aminohexanoic acid) and with TMR (tetramethylrhodamine). PB-M7VIS is efficiently and selectively cleaved by MMP-7 with a k (cat)/ K (m) value of 1.9x10(5) M(-1).s(-1) as measured by the rate of increase in Fl fluorescence (up to 17-fold for the cleavage of an optimized PB-M7VIS) with minimal change in the TMR fluorescence. The K (m) value for PB-M7VIS is approx. 0.5 microM, which is approx. two orders of magnitude lower when compared with that for an analogous soluble peptide, indicating efficient interaction of MMP-7 with the synthetic polymeric substrate. With MMP-2 or -3, the k (cat)/ K (m) value for PB-M7VIS is approx. 56- or 13-fold lower respectively, when compared with MMP-7. In PB-M7VIS, Fl(AHX)RPLALWRS(AHX)C is a selective optical sensor of MMP-7 activity and TMR serves to detect both the uncleaved and cleaved reagents. Each of these can be visualized as subcutaneous fluorescent phantoms in a mouse and optically discriminated based on the ratio of green/red (Fl/TMR) fluorescence. The in vivo specificity of PB-M7VIS was tested in a mouse xenograft model. Intravenous administration of PB-M7VIS gave significantly enhanced Fl fluorescence from MMP-7-positive tumours, but not from control tumours ( P <0.0001), both originally derived from SW480 human colon cancer cells. Prior systemic treatment of the tumour-bearing mice with an MMP inhibitor BB-94 ([4-( N -hydroxyamino)-2 R -isobutyl-3 S -(thienylthiomethyl)-succinyl]-L-phenylalanine- N -methylamide), markedly decreased the Fl fluorescence over the MMP-7-positive tumour by approx. 60%. Thus PB-M7VIS functions as a PB for in vivo detection of MMP-7 activity that serves to light this optical beacon and is, therefore, a selective in vivo optical molecular imaging contrast reagent.

The adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine (A to I) in primary mRNA transcripts, thereby affecting the splicing pattern or coding potential of mature mRNAs. Although the subnuclear localization of A-to-I editing has not been precisely defined, ADARs have been shown to act before splicing, suggesting that they function near nucleoplasmic sites of transcription. Here we demonstrate that ADAR2, a member of the vertebrate ADAR family, is concentrated in the nucleolus, a subnuclear domain disparate from the sites of mRNA transcription. Selective inhibition of ribosomal RNA synthesis or the introduction of mutations in the double-stranded RNA-binding domains within ADAR2 results in translocation of the protein to the nucleoplasm, suggesting that nucleolar association of ADAR2 depends on its ability to bind to ribosomal RNA. Fluorescence recovery after photobleaching reveals that ADAR2 can shuttle rapidly between subnuclear compartments. Enhanced translocation of endogenous ADAR2 from the nucleolus to the nucleoplasm results in increased editing of endogenous ADAR2 substrates. These observations indicate that the nucleolar localization of ADAR2 represents an important mechanism by which RNA editing can be modulated by the sequestration of enzymatic activity from potential RNA substrates in the nucleoplasm.

Objective: Co-expression of the KvLQT1 and minK potassium channel subunits is required to recapitulate I(Ks), the slow component of the cardiac delayed rectifier current, and mutations in either gene cause the congenital Long QT syndrome. It is becoming increasingly well-recognized that multiprotein channel complexes containing proteins capable of modulating channel function assemble at the plasma membrane. Thus, the aim of our study was to identify proteins involved in I(Ks) modulation., Methods and Results: Using a yeast-two-hybrid screen with the intracytoplasmic C-terminus of minK as bait, we identified the cardiac-enriched four-and-a-half LIM domain-containing protein (fhl2) as a potential minK partner. We show interaction between the two proteins in GST pulldown assays and demonstrate overlapping subcellular localization using immunocytochemistry of transfected cells supporting a potential interaction. At the functional level, expression of KvLQT1and minK in HEK cells, which lack endogenous fhl2 protein, generated I(Ks) only when fhl2 was co-expressed. By contrast, in CHO-K1 cells, which express fhl2 endogenously, I(Ks) was suppressed by anti-fhl2 antisense which did not affect the currents generated by KvLQT1alone., Conclusion: These data indicate that at least in heterologous cells, the generation of I(Ks) requires fhl2 as an additional protein component.

A new series of high-affinity cyanine dyes was tested for the visualization of the dynamics of single DNA molecules through a fluorescence microscope. In particular, YOYO-1 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)- bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)- 2-methylidene]-quinolinium tetraiodide) forms a very stable, highly fluorescent complex with double-stranded DNA and dramatically improves the quality of the images. We have characterized the factors affecting the signal/background in the imaging of single DNA molecules by fluorescence microscopy and compared the results obtained using YOYO-1 with those obtained using standard fluorescent dyes like ethidium bromide or acridine orange.

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

Pulsed field gel electrophoresis (PFGE) techniques have been developed to overcome the limitations of conventional electrophoresis and to increase the separation to DNA chromosomes of few megabase pairs in size. Despite of the large success of these techniques, the various separation protocols employed for PFGE experiments have been determined empirically. However, a deep understanding of the molecular mechanisms of motion responsible for DNA separation becomes necessary for the rational optimization of these techniques. This paper shows the first clear observations of individual molecules of DNA during the reorientation process in 90 degrees PFGE and 120 degrees PFGE. Real-time visualization of the DNA dynamics during PFGE was possible with the use of an epi-illumination fluorescence microscope specifically equipped to run these experiments and by staining the DNA with YOYO-1 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-meth yl -2,3-dihydro-(benzo-1,3-oxazole)-2-methyl-idene]-quinolinium tetraiodide). This dye forms a very stable, highly fluorescent complex with double-stranded DNA and dramatically improves the quality of the DNA images. The results of computer simulations used to reproduce the molecular mechanisms of motion as well as the DNA separation features are also discussed.

We used the phosphatase substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6- chloro-4-[3H]-quinazolinone, with standard alkaline phosphatase-mediated immunohistochemical techniques, to visualize a number of antibodies that bind to adult zebrafish retinal tissue. This compound, known as the ELF (enzyme-labeled-fluorescence) phosphatase substrate, produces a precipitate that fluoresces at approximately 500-580 nm (bright yellow-green). We show that the precipitated product from the ELF phosphatase substrate has a number of characteristics that make it superior to fluorescein-labeled secondary reagents. The staining produced with the ELF substrate is much more photostable than that produced by fluorescein-labeled secondary reagents, thus allowing time to examine, focus, and photograph the ELF-labeled tissue under high magnification. Moreover, the ELF precipitate exhibits a Stokes shift of greater than 100 nm, a characteristic that has enabled us to overcome the problem of distinguishing signal from background in this autofluorescent tissue. In addition, we show that the ELF product's large Stokes shift makes the ELF substrate ideal for multicolor applications.

The ELF alkaline phosphate substrate can be used to fluorescently label a wide variety of biological targets. This substrate yields a bright, photostable yellow-green fluorescent precipitate at the site of enzymatic activity. ELF labeling can be as much as 40 times as bright and hundreds of times as photostable as labeling with conventional fluorophores and yields signals capable of very fine submicroscopic resolution. Signal development is also extremely rapid, making the signal amplification technology well suited for applications such as RNA in situ hybridization.

We developed site-specific fluorescent probes that permit simultaneous microscopic observation of G- and F-actin in bovine endothelial cells. G-actin distribution was visualized with fluorescein-deoxyribonuclease I (DNAse I). F-actin was labeled with phalloidin conjugated to the new long-wavelength fluorophore BODIPY 581/591 (581-nm excitation, 591-nm emission), which is spectrally similar to Texas Red. The G-actin appeared as pervasive green fluorescence that was more intense in the nuclear region, where cell thickness is greater and stress fibers are less frequent. In addition, we observed a punctate fluorescein pattern around the nuclei and in other parts of the cells, suggesting that some G-actin is localized to small discrete sites. F-actin was observed as red fluorescent filaments. Unlabeled DNAse I effectively prevented staining of G-actin by the fluorescent DNAse I conjugates. The specificity of DNAse I for G-actin was confirmed by the presence of a single labeled band with molecular weight corresponding to actin in a Western blot of total cytoplasmic endothelial proteins reacted with biotin-DNAse I-streptavidin-alkaline phosphatase. Anti-actin antibody, which associates with both G- and F-actin, in conjunction with fluorescent secondary antibody produced a pattern similar to that obtained by simultaneous visualization with fluorescein-DNAse I and BODIPY 581/591- or rhodamine-phalloidin.

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In this paper we test the predictions of the differential polarization imaging theory developed in the previous two papers. A characterization of the patterns of polymerization of hemoglobin in red blood cells from patients with sickle cell anemia is presented. This system was chosen because it is relatively easy to handle and because previous studies have been done on it. A differential polarization microscope designed and built in our laboratory was used to carry out this study. This microscope uses an image dissector camera, a photoelastic modulator, and a phase-lock amplifier. This design represents a substantial modification with respect to the instrumentation used in the previous results communicated on this system. Therefore, the results presented here also permit us to confirm the validity of our conclusions. On the basis of the differential polarization images obtained, models of the patterns of polymerization of the hemoglobin S inside the sickle cells are proposed and their M12 and regular images are calculated by the theory. Good agreement between those models and the experimental systems is found, as well as with the results previously reported.

In this paper we test the predictions of the differential polarization imaging theory developed in the previous two papers. A characterization of the patterns of polymerization of hemoglobin in red blood cells from patients with sickle cell anemia is presented. This system was chosen because it is relatively easy to handle and because previous studies have been done on it. A differential polarization microscope designed and built in our laboratory was used to carry out this study. This microscope uses an image dissector camera, a photoelastic modulator, and a phase-lock amplifier. This design represents a substantial modification with respect to the instrumentation used in the previous results communicated on this system. Therefore, the results presented here also permit us to confirm the validity of our conclusions. On the basis of the differential polarization images obtained, models of the patterns of polymerization of the hemoglobin S inside the sickle cells are proposed and their M12 and regular images are calculated by the theory. Good agreement between those models and the experimental systems is found, as well as with the results previously reported.