Measurement of Isolated Myocyte Volume Using the Coulter
            <sup>®</sup>
            Models Z2 and ZM/C256: A Comparison of Instrument Function

Said, Suleman; Tamura, Tetsutaro; Gerdes, A. Martin

doi:10.2144/98253pf02

Cited by 7 publications

(7 citation statements)

References 14 publications

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“…Furthermore, cellular volumes of myocytes isolated from PKC⑀ KO hearts were no different from wild-type cardiac myocytes (22,275 Ϯ 958 versus 24,565 Ϯ 918 m 3 ; n ϭ 4, p ϭ not significant) as measured electronically with a Coulter counter (22). Therefore, targeted disruption of the PKC⑀ gene had no obvious effect on overall heart morphology or individual cardiac myocyte size.…”

Section: Normal Base-line Morphology and In Vivo Cardiac Function In mentioning

confidence: 84%

Preservation of Base-line Hemodynamic Function and Loss of Inducible Cardioprotection in Adult Mice Lacking Protein Kinase Cϵ

Gray

Zhou

Schafhalter-Zoppoth

et al. 2004

Journal of Biological Chemistry

104

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Signaling pathways involving protein kinase C isozymes are modulators of cardiovascular development and response to injury. Protein kinase C⑀ activation in cardiac myocytes reduces necrosis caused by coronary artery disease. However, it is unclear whether protein kinase C⑀ function is required for normal cardiac development or inducible protection against oxidative stress. Protein kinase C␦ activation is also observed during cardiac preconditioning. However, its role as a promoter or inhibitor of injury is controversial. We examined hearts from protein kinase C⑀ knock-out mice under physiological conditions and during acute ischemia reperfusion. Null-mutant and wild-type mice displayed equivalent base-line morphology and hemodynamic function. Targeted disruption of the protein kinase C⑀ gene blocked cardioprotection caused by ischemic preconditioning and ␣ 1 -adrenergic receptor stimulation. Protein kinase C␦ activation increased in protein kinase C⑀ knock-out myocytes without altering resistance to injury. These observations support protein kinase C⑀ activation as an essential component of cardioprotective signaling. Our results favor protein kinase C␦ activation as a mediator of normal growth. This study advances the understanding of cellular mechanisms responsible for preservation of myocardial integrity as potential targets for prevention and treatment of ischemic heart disease.Coronary artery disease and chronic heart failure are important causes of death worldwide. Cellular signaling pathways that regulate cardiovascular development and responses to injury, particularly those involving protein kinase C (PKC) 1 isozymes, are under intense investigation. For example, MochlyRosen et al. (1) found that activation of PKC⑀ translocation in transgenic mouse hearts caused physiological hypertrophy and reduced the size of individual myocytes. In contrast, postnatal inhibition of PKC⑀ translocation produced lethal dilated cardiomyopathy and increased cardiac myocyte volumes (1). Wu et al. (2) later observed that activation of PKC⑀ translocation in G␣ q transgenic hearts improved contractile function. Conversely, inhibition of PKC⑀ translocation converted the G␣ q hypertrophic phenotype to a dilated cardiomyopathy (2). However, no previous investigations established whether PKC⑀ activation was an absolute requirement for normal cardiac growth or whether compensatory changes in the expression of other myocardial proteins developed in the absence of PKC⑀-mediated signaling.Two lines of evidence support the hypothesis that PKC⑀ activation promotes myocardial resistance to injury during periods of oxidative stress. First, both ischemic preconditioning and pharmacological approaches that induce cardioprotection increase PKC⑀ immunoreactivity and kinase function in cell particulate fractions (3). Second, transgenic expression of a constitutively active PKC⑀ (4) or a peptide agonist of PKC⑀ translocation (5) reduces cardiac myocyte necrosis during ischemia reperfusion. However, it is unclear whether PKC⑀ activation represe...

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Section: Normal Base-line Morphology and In Vivo Cardiac Function In mentioning

confidence: 84%

Preservation of Base-line Hemodynamic Function and Loss of Inducible Cardioprotection in Adult Mice Lacking Protein Kinase Cϵ

Gray

Zhou

Schafhalter-Zoppoth

et al. 2004

Journal of Biological Chemistry

104

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“…To examine whether the increase in FSC observed in E-myc mice actually corresponds to an increase in cell volume, we purified B lymphocytes from young E-myc transgenic mice and analyzed cell volume via the Coulter principle (47). As shown in Fig.…”

Section: C-myc Transgene Expression Results In Increased Growth Of Prmentioning

confidence: 99%

“…Cell volume measurements were performed by using a Coulter Model Z2 (Coulter) (47). Cells were diluted in Isoton II (Beckman Coulter) at 100,000 cells͞ml in 10 ml.…”

Section: Methodsmentioning

confidence: 99%

c-Myc enhances protein synthesis and cell size during B lymphocyte development

Iritani

Eisenman

1999

Proc. Natl. Acad. Sci. U.S.A.

330

223

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It has been known for some time that cell cycle progression is tightly coupled to an accumulation of cell mass (i.e., cell growth) (1). However, the molecules that control cell growth and the mechanisms through which growth and proliferation are coupled are only beginning to be defined (2). One group of genes whose function may be important for both growth and proliferation is the members of the myc protooncogene family, c-, N-, and L-myc.The proteins encoded by myc family genes are members of the basic-helix-loop-helix-zipper (bHLHZ) class of transcription factors. Dimerization of Myc protein with its obligate partner Max results in formation of a heterodimer with sequence-specific DNA-binding activity (3, 4). Myc-Max heterodimers appear to activate transcription when bound to promoter-proximal sites on DNA (5, 6). In addition, Myc is known to repress transcription of specific genes (7). Although max expression is constitutive, myc expression is highly regulated at transcriptional, posttranscriptional, translational, and posttranslational levels (8-11). In general, c-Myc expression is associated with proliferation and is down-regulated in quiescent and differentiated cells. After serum or mitogen stimulation of quiescent cells, myc levels peak within several hours, followed by a decline to a low basal level maintained by synthesis and degradation (12-15) and dependent on the continued presence of growth factors (16). Ectopic expression of c-myc in normally quiescent cells can potentiate entry into S phase, and cells that constitutively express myc have reduced growth factor requirements, shortened doubling times, and in some cases have circumvented cell cycle exit (17)(18)(19)(20). Conversely, failure to induce c-myc in response to mitogenic signaling abrogates cell cycle progression (21) whereas a fibroblast cell line bearing targeted homozygous c-myc deletions has significantly decreased proliferation rates and lengthened G 1 and G 2 phases (22). These observations together have led to the notion that Myc-Max functions to modulate expression of genes promoting cell cycle progression (for reviews, see refs. 23-25).Deregulated expression of myc family genes, through gene amplification, viral promoter insertion, chromosomal translocation, or promoter mutation, has long been associated with neoplastic disease in a wide range of vertebrates including humans (for reviews, see refs. 26-29). Among the most striking examples of the importance of myc in cancer development are the chromosomal translocations involving c-myc and Ig heavy-or light-chain loci that are characteristic of Burkitt's lymphoma in humans and plasmacytomas in mice and rats. A powerful model system for B cell lymphoma (E-myc mice) entails expression of c-myc in murine lymphoid cells as a transgene under control of the Ig heavy-chain enhancer (30) (for review see ref. 31). E-myc mice develop clonal B cell lymphomas with a mean latency of 12-16 wk of age. Before transformation, B cell progenitors appear relatively normal in these mice. However, an exp...

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“…Three-dimensional measurements of isolated NRVMs were conducted using a Coulter model Z2 instrument (Beckman), as previously described (54). Briefly, cells in six-well dishes (2.5 ϫ 10 5 /well) were washed one time with PBS and trypsinized (200 l trypsin, 2 min at room temperature).…”

Section: Methodsmentioning

confidence: 99%

The CRM1 Nuclear Export Receptor Controls Pathological Cardiac Gene Expression

Harrison¹,

Roberts²,

Hood³

et al. 2004

Molecular and Cellular Biology

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Diverse pathological insults trigger a cardiac remodeling process during which myocytes undergo hypertrophy, with consequent decline in cardiac function and eventual heart failure. Multiple transcriptional regulators of pathological cardiac hypertrophy are controlled at the level of subcellular distribution. For example, prohypertrophic transcription factors belonging to the nuclear factor of activated T cells (NFAT) and GATA families are subject to CRM1-dependent nuclear export but are rapidly relocalized to the nucleus in response to cues for hypertrophic growth. Here, we demonstrate that the antihypertrophic chromatin-modifying enzyme histone deacetylase 5 (HDAC5) is shuttled out of the cardiomyocyte nucleus via a CRM1-mediated pathway in response to diverse signals for hypertrophy. CRM1 antagonists block the agonist-mediated nuclear export of HDAC 5 and repress pathological gene expression and associated hypertrophy of cultured cardiomyocytes. Conversely, CRM1 activity is dispensable for nonpathological cardiac gene activation mediated by thyroid hormone and insulin-like growth factor 1, agonists that fail to trigger the nuclear export of HDAC5. These results suggest a selective role for CRM1 in derepression of pathological cardiac genes via its neutralizing effects on antihypertrophic factors such as HDAC5. Pharmacological approaches targeting CRM1-dependent nuclear export in heart muscle may have salutary effects on cardiac function by suppressing maladaptive changes in gene expression evoked by stress signals.A common mechanism controlling gene expression involves altering the subcellular distribution of transcriptional regulators. A multitude of transcription factors and cofactors possess nuclear localization sequences (NLSs) and nuclear export signals (NESs) that mediate entry into and exit from the nucleus, respectively. Frequently, signal transduction pathways that impinge on transcriptional regulators function by positively or negatively affecting the activities of these intrinsic targeting domains.For proteins over ϳ40 kDa, passage into and out of the nucleus is governed by the nuclear pore complex (NPC), a multisubunit structure embedded in the nuclear envelope (27). Positively charged NLSs are bound by importins ␣ and ␤, which tether cargo to the cytosolic face of the NPC and facilitate translocation of proteins into the nucleus. The CRM1 protein, also referred to as exportin, mediates the transit of proteins out of the nucleus (16), although CRM1-independent mechanisms for nuclear export exist (25, 33). CRM1 binds hydrophobic NESs together with the small GTP binding protein Ran, and these ternary complexes are shuttled out of the nucleus through a series of interactions with the NPC.

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Measurement of Isolated Myocyte Volume Using the Coulter ^® Models Z2 and ZM/C256: A Comparison of Instrument Function

Cited by 7 publications

References 14 publications

Preservation of Base-line Hemodynamic Function and Loss of Inducible Cardioprotection in Adult Mice Lacking Protein Kinase Cϵ

Preservation of Base-line Hemodynamic Function and Loss of Inducible Cardioprotection in Adult Mice Lacking Protein Kinase Cϵ

c-Myc enhances protein synthesis and cell size during B lymphocyte development

The CRM1 Nuclear Export Receptor Controls Pathological Cardiac Gene Expression

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Measurement of Isolated Myocyte Volume Using the Coulter ® Models Z2 and ZM/C256: A Comparison of Instrument Function

Cited by 7 publications

References 14 publications

Preservation of Base-line Hemodynamic Function and Loss of Inducible Cardioprotection in Adult Mice Lacking Protein Kinase Cϵ

Preservation of Base-line Hemodynamic Function and Loss of Inducible Cardioprotection in Adult Mice Lacking Protein Kinase Cϵ

c-Myc enhances protein synthesis and cell size during B lymphocyte development

The CRM1 Nuclear Export Receptor Controls Pathological Cardiac Gene Expression

Contact Info

Product

Resources

About

Measurement of Isolated Myocyte Volume Using the Coulter ^® Models Z2 and ZM/C256: A Comparison of Instrument Function