A Modified Density Gradient Proteomic-Based Method to Analyse Endolysosomal Proteins in Cardiac Tissue

SummaryThe importance of lysosomes in cardiac physiology and pathology are well established, and evidence for roles in calcium signalling are emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endo-lysosomal (EL) proteins.Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/ml); and EL with negligible contamination from SR or Mito (1.04 g/ml), were analysed using Western Blot, enzyme activity assay and LC-MS/MS analysis (adapted discontinuous Percoll, and sucrose differential density gradient).Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and dataset of guinea-pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

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Activated Prominences; Mechanisms for Activation and Eruption

International Astronomical Union Colloquium ◽

10.1017/s0252921100065714 ◽

1979 ◽

Vol 44 ◽

pp. 307-313

Author(s):

D.S. Spicer

Keyword(s):

Pressure Gradient ◽

Density Gradient ◽

Current Density ◽

Convective Instability ◽

Tearing Mode ◽

Magnetic Shear ◽

Long Wavelength ◽

Ideal Mhd ◽

Gradient Change ◽

Accelerated Particles

A possible relationship between the hot prominence transition sheath, increased internal turbulent and/or helical motion prior to prominence eruption and the prominence eruption (“disparition brusque”) is discussed. The associated darkening of the filament or brightening of the prominence is interpreted as a change in the prominence’s internal pressure gradient which, if of the correct sign, can lead to short wavelength turbulent convection within the prominence. Associated with such a pressure gradient change may be the alteration of the current density gradient within the prominence. Such a change in the current density gradient may also be due to the relative motion of the neighbouring plages thereby increasing the magnetic shear within the prominence, i.e., steepening the current density gradient. Depending on the magnitude of the current density gradient, i.e., magnetic shear, disruption of the prominence can occur by either a long wavelength ideal MHD helical (“kink”) convective instability and/or a long wavelength resistive helical (“kink”) convective instability (tearing mode). The long wavelength ideal MHD helical instability will lead to helical rotation and thus unwinding due to diamagnetic effects and plasma ejections due to convection. The long wavelength resistive helical instability will lead to both unwinding and plasma ejections, but also to accelerated plasma flow, long wavelength magnetic field filamentation, accelerated particles and long wavelength heating internal to the prominence.

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Cytopathology of Cardiac Tissue from Daunomycin-Treated Mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066802 ◽

1971 ◽

Vol 29 ◽

pp. 550-551

Author(s):

Robert H. Liss ◽

Frances A. Cotton

Keyword(s):

Cardiac Tissue ◽

Cardiac Toxicity ◽

Drug Distribution ◽

Personal Communication ◽

Intravenous Dose ◽

Single Intravenous Dose ◽

Physiologic Saline ◽

Histopathologic Changes ◽

Distribution Studies ◽

Lung And Kidney

Daunomycin, an antibiotic used in the clinical management of acute leukemia, produces a delayed, lethal cardiac toxicity. The lethality is dose and schedule dependent; histopathologic changes induced by the drug have been described in heart, lung, and kidney from hamsters in both single and multiple dose studies. Mice given a single intravenous dose of daunomycin (10 mg/kg) die 6-7 days later. Drug distribution studies indicate that the rodents excrete most of a single dose of the drug as daunomycin and metabolite within 48 hours after dosage (M. A. Asbell, personal communication).Myocardium from the ventricles of 6 moribund BDF1 mice which had received a single intravenous dose of daunomycin (10 mg/kg), and from controls dosed with physiologic saline, was fixed in glutaraldehyde and prepared for electron microscopy.

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Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100064037 ◽

1969 ◽

Vol 27 ◽

pp. 424-425

Author(s):

Lee F. Ellis ◽

Richard M. Van Frank ◽

Walter J. Kleinschmidt

Keyword(s):

Electron Microscopy ◽

Tissue Culture ◽

Density Gradient ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Uranyl Acetate ◽

Interferon Inducer ◽

Virus Particles ◽

Staining Methods ◽

Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

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Iron storage sites in the myocardium as revealed by cytohistochemistry

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100104030 ◽

1988 ◽

Vol 46 ◽

pp. 394-395

Author(s):

M. Ashraf ◽

F. Thompson ◽

S. Miki ◽

P. Srivastava

Keyword(s):

Cardiac Tissue ◽

Coronary Perfusion ◽

Intracellular Iron ◽

Ether Anesthesia ◽

Intense Staining ◽

Iron Storage ◽

Thin Sections ◽

Rat Hearts ◽

Cacodylate Buffer ◽

Made In

Iron is believed to play an important role in the pathogenesis of ischemic injury. However, the sources of intracellular iron in myocytes are not yet defined. In this study we have attempted to localize iron at various cellular sites of the cardiac tissue with the ferrocyanide technique.Rat hearts were excised under ether anesthesia. They were fixed with coronary perfusion with 3% buffered glutaraldehyde made in 0.1 M cacodylate buffer pH 7.3. Sections, 60 μm in thickness, were cut on a vibratome and were incubated in the medium containing 500 mg of potassium ferrocyanide in 49.5 ml H2O and 0.5 ml concentrated HC1 for 30 minutes at room temperature. Following rinses in the buffer, tissues were dehydrated in ethanol and embedded in Spurr medium.The examination of thin sections revealed intense staining or reaction product in peroxisomes (Fig. 1).

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Immunoelectron microscope detection of C-type natriuretic peptide in normal human atrial tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147776 ◽

1993 ◽

Vol 51 ◽

pp. 388-389

Author(s):

Chi-Ming Wei ◽

Margaret Hukee ◽

Christopher G.A. McGregor ◽

John C. Burnett

Keyword(s):

Amino Acid ◽

Natriuretic Peptide ◽

Vascular Tone ◽

Cardiac Tissue ◽

Ring Structure ◽

Terminal Amino Acid ◽

Amino Acid Peptide ◽

Normal Human ◽

Terminal Amino ◽

The Brain

C-type natriuretic peptide (CNP) is a newly identified peptide that is structurally related to atrial (ANP) and brain natriuretic peptide (BNP). CNP exists as a 22-amino acid peptide and like ANP and BNP has a 17-amino acid ring formed by a disulfide bond. Unlike these two previously identified cardiac peptides, CNP lacks the COOH-terminal amino acid extension from the ring structure. ANP, BNP and CNP decrease cardiac preload, but unlike ANP and BNP, CNP is not natriuretic. While ANP and BNP have been localized to the heart, recent investigations have failed to detect CNP mRNA in the myocardium although small concentrations of CNP are detectable in the porcine myocardium. While originally localized to the brain, recent investigations have localized CNP to endothelial cells consistent with a paracrine role for CNP in the control of vascular tone. While CNP has been detected in cardiac tissue by radioimmunoassay, no studies have demonstrated CNP localization in normal human heart by immunoelectron microscopy.

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Confocal imaging of calcium in intact ventricular muscle provides a new view of excitation-contraction coupling

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166154 ◽

1996 ◽

Vol 54 ◽

pp. 738-739

Author(s):

W.G. Wier

Keyword(s):

Local Control ◽

Cardiac Tissue ◽

Cell Function ◽

Isolated Heart ◽

Cardiac Cells ◽

Confocal Imaging ◽

Ion Concentration ◽

Heart Cell ◽

Free Calcium ◽

Heart Cells

A fundamentally new understanding of cardiac excitation-contraction (E-C) coupling is being developed from recent experimental work using confocal microscopy of single isolated heart cells. In particular, the transient change in intracellular free calcium ion concentration ([Ca2+]i transient) that activates muscle contraction is now viewed as resulting from the spatial and temporal summation of small (∼ 8 μm3), subcellular, stereotyped ‘local [Ca2+]i-transients' or, as they have been called, ‘calcium sparks'. This new understanding may be called ‘local control of E-C coupling'. The relevance to normal heart cell function of ‘local control, theory and the recent confocal data on spontaneous Ca2+ ‘sparks', and on electrically evoked local [Ca2+]i-transients has been unknown however, because the previous studies were all conducted on slack, internally perfused, single, enzymatically dissociated cardiac cells, at room temperature, usually with Cs+ replacing K+, and often in the presence of Ca2-channel blockers. The present work was undertaken to establish whether or not the concepts derived from these studies are in fact relevant to normal cardiac tissue under physiological conditions, by attempting to record local [Ca2+]i-transients, sparks (and Ca2+ waves) in intact, multi-cellular cardiac tissue.

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