Characterization of a heat shock-induced insoluble complex in the nuclei of cells

The formation of an insoluble complex in isolated nuclei incubated at physiological temperature (37 degrees C) is demonstrated. A similar complex is shown to form in the nuclei of intact cells subjected to temperatures that induce the classical heat-shock response. The formation of this complex occurs rapidly in response to hyperthermia and is induced by small increases in temperature both in vitro and in vivo. We have characterized the formation of the complex in isolated nuclei and the nuclei of intact cells. A small number of the subset of nuclear proteins involved in the complex have been identified. The significance of the loss of solubility of these proteins in the nucleus following hyperthermia is discussed.

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Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60

Biochemical Journal ◽

10.1042/bj20050861 ◽

2005 ◽

Vol 391 (2) ◽

pp. 185-190 ◽

Cited By ~ 64

Author(s):

Renu Wadhwa ◽

Syuichi Takano ◽

Kamaljit Kaur ◽

Satoshi Aida ◽

Tomoko Yaguchi ◽

...

Keyword(s):

Heat Shock ◽

Molecular Interactions ◽

Heat Shock Protein 60 ◽

Hsp60 Expression ◽

Mitochondrial Hsp70 ◽

Shock Proteins ◽

First Time

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

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Characterization of Grb4, an adapter protein interacting with Bcr-Abl

Blood ◽

10.1182/blood.v96.2.618.014k06_618_624 ◽

2000 ◽

Vol 96 (2) ◽

pp. 618-624 ◽

Cited By ~ 3

Author(s):

Sunita Coutinho ◽

Thomas Jahn ◽

Marc Lewitzky ◽

Stephan Feller ◽

Peter Hutzler ◽

...

Keyword(s):

Myelogenous Leukemia ◽

Adapter Protein ◽

Intact Cells ◽

Abl Tyrosine Kinase ◽

Abl Kinases ◽

Src Homology ◽

Dependent Interaction

We report here the characterization of an adapter protein identified in a yeast 2-hybrid screen with the use of Bcr-Abl as the bait. Grb4 bound to Bcr-Abl in a variety of systems, both in vitro and in vivo, and is an excellent substrate of the Bcr-Abl tyrosine kinase. The association of Grb4 and Bcr-Abl in intact cells was mediated by an src homology (SH)2–mediated phosphotyrosine-dependent interaction as well as an SH3-mediated phosphotyrosine-independent interaction. Grb4 has 68% homology to the adapter protein Nck and has similar but distinct binding specificities in K562 lysates. Subcellular localization studies indicate that Grb4 localizes to both the nucleus and the cytoplasm. Coexpression of kinase-active Bcr-Abl with Grb4 resulted in the translocation of Grb4 from the cytoplasm and the nucleus to the cytoskeleton to colocalize with Bcr-Abl. In addition, expression of Grb4 with kinase-active Bcr-Abl resulted in a redistribution of actin-associated Bcr-Abl. Finally, coexpression of Grb4 and oncogenic v-Abl strongly inhibited v-Abl–induced AP-1 activation. Together, these data indicate that Grb4 in conjunction with Bcr-Abl may be capable of modulating the cytoskeletal structure and negatively interfering with the signaling of oncogenic Abl kinases. Grb4 may therefore play a role in the molecular pathogenesis of chronic myelogenous leukemia. (Blood. 2000;96:618-624)

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Physiological requirements for ciliary reactivation of bracken fern spermatozoids

Journal of Cell Science ◽

10.1242/jcs.43.1.195 ◽

1980 ◽

Vol 43 (1) ◽

pp. 195-207

Author(s):

S.M. Wolniak ◽

W.Z. Cande

Keyword(s):

Cell Model ◽

Pteridium Aquilinum ◽

Free Calcium ◽

Permeabilized Cell ◽

Intact Cells ◽

Cell Model System ◽

Ciliary Beat

Physiological parameters affecting reactivated ciliary beat in spermatozoids of braken fern (Pteridium aquilinum) were studied using a Triton/glycerol permeabilized cell model system. Reactivation frequencies of polylysine-tethered cells equalled in vivo rates at neutral pH. Frequency was dependent on ATP and Mg2+ concentration, and reactivation was inhibited by millimolar or greater free calcium. Reactivation was reversibly inhibited by micromolar concentrations of sodium ortho-vanadate, while intact cells were not affected by millimolar levels of the inhibitor. This is the first characterization of in vitro ciliary beat in a non-algal plant cell and demonstrates that the nucleotide and ionic requirements for reactivation of bracken cilia are similar to those of other systems.

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Characterization of the small molecule ARC39, a direct and specific inhibitor of acid sphingomyelinase in vitro

The Journal of Lipid Research ◽

10.1194/jlr.ra120000682 ◽

2020 ◽

Vol 61 (6) ◽

pp. 896-910 ◽

Cited By ~ 5

Author(s):

Eyad Naser ◽

Stephanie Kadow ◽

Fabian Schumacher ◽

Zainelabdeen H. Mohamed ◽

Christian Kappe ◽

...

Keyword(s):

Cultured Cells ◽

Specific Inhibitor ◽

Acid Sphingomyelinase ◽

Intact Cells ◽

Protein Levels ◽

High Doses ◽

Hydrolysis Of

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM’s catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

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Caloric restriction induces heat shock response and inhibits B16F10 cell tumorigenesis both in vitro and in vivo

Aging ◽

10.18632/aging.100732 ◽

2015 ◽

Vol 7 (4) ◽

pp. 233-240 ◽

Cited By ~ 3

Author(s):

Marta G. Novelle ◽

Ashley Davis ◽

Nathan L. Price ◽

Ahmed Ali ◽

Stefanie Fürer-Galvan ◽

...

Keyword(s):

Heat Shock ◽

Caloric Restriction ◽

Heat Shock Response ◽

B16f10 Cell ◽

Shock Response

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Characterization of a recombinant porcine follistatin in a heat shock expression system

Canadian Journal of Animal Science ◽

10.4141/a01-056 ◽

2002 ◽

Vol 82 (3) ◽

pp. 295-304

Author(s):

C. R. Christensen ◽

J. Kowalski ◽

P. J. Chedrese ◽

V. Misra ◽

B. Laarveld ◽

...

Keyword(s):

Heat Shock ◽

Ovarian Function ◽

Expression System ◽

Porcine Granulosa Cells ◽

Molecular Masses ◽

Estradiol 17Β

The role of follistatin in ovarian function has yet to be fully elucidated; it is present in low concentration in vivo and it is difficult to obtain suitable quantities of follistatin for characterization. We have cloned porcine follistatin cDNA in an expression system that uses the heat shock protein promoter BoHSP70. Recombinant follistatin with apparent molecular masses of 39, 46, 48, 50 kDa was expressed and secreted into culture medium at concentrations of 400–500 μg × 20 mL-1 × 150 cm-2 flask (4 × 107 cells). In ligand blots, the recombinant follistatin was shown to be immunologically similar to native follistatin and to bind to recombinant activin A. Porcine granulosa cells dissected from 1–3 mm follicles from immature gilts were cultured with recombinant follistatin or with a follistatin monoclonal antibody to examine the activity of the recombinant follistatin. At a physiologically relevant concentration of 1 μg mL-1 the recombinant porcine follistatin suppressed the accumulation of estradiol-17β (P < 0.05). There was a trend for estradiol-17β accumulation in the presence of 10 μg mL-1 of monoclonal anti-follistatin antibody (P = 0.1). This expression system consistently produced large quantities of recombinant porcine follistatin, which is immunologically and biologically similar to follistatin, and is capable of independently inhibiting estratiol-17βproduction in vitro. Key words: Porcine, follistatin, heat shock promoter, glycoprotein, ovary, estradiol

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Characterization of Grb4, an adapter protein interacting with Bcr-Abl

Blood ◽

10.1182/blood.v96.2.618 ◽

2000 ◽

Vol 96 (2) ◽

pp. 618-624 ◽

Cited By ~ 18

Author(s):

Sunita Coutinho ◽

Thomas Jahn ◽

Marc Lewitzky ◽

Stephan Feller ◽

Peter Hutzler ◽

...

Keyword(s):

Myelogenous Leukemia ◽

Adapter Protein ◽

Intact Cells ◽

Abl Tyrosine Kinase ◽

Abl Kinases ◽

Src Homology ◽

Dependent Interaction

Abstract We report here the characterization of an adapter protein identified in a yeast 2-hybrid screen with the use of Bcr-Abl as the bait. Grb4 bound to Bcr-Abl in a variety of systems, both in vitro and in vivo, and is an excellent substrate of the Bcr-Abl tyrosine kinase. The association of Grb4 and Bcr-Abl in intact cells was mediated by an src homology (SH)2–mediated phosphotyrosine-dependent interaction as well as an SH3-mediated phosphotyrosine-independent interaction. Grb4 has 68% homology to the adapter protein Nck and has similar but distinct binding specificities in K562 lysates. Subcellular localization studies indicate that Grb4 localizes to both the nucleus and the cytoplasm. Coexpression of kinase-active Bcr-Abl with Grb4 resulted in the translocation of Grb4 from the cytoplasm and the nucleus to the cytoskeleton to colocalize with Bcr-Abl. In addition, expression of Grb4 with kinase-active Bcr-Abl resulted in a redistribution of actin-associated Bcr-Abl. Finally, coexpression of Grb4 and oncogenic v-Abl strongly inhibited v-Abl–induced AP-1 activation. Together, these data indicate that Grb4 in conjunction with Bcr-Abl may be capable of modulating the cytoskeletal structure and negatively interfering with the signaling of oncogenic Abl kinases. Grb4 may therefore play a role in the molecular pathogenesis of chronic myelogenous leukemia. (Blood. 2000;96:618-624)

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Histamine Is an Inducer of the Heat Shock Response in SOD1-G93A Models of ALS

International Journal of Molecular Sciences ◽

10.3390/ijms20153793 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3793 ◽

Cited By ~ 2

Author(s):

Savina Apolloni ◽

Francesca Caputi ◽

Annabella Pignataro ◽

Susanna Amadio ◽

Paola Fabbrizio ◽

...

Keyword(s):

Heat Shock ◽

Motor Neuron ◽

Motor Neurons ◽

Heat Shock Response ◽

Type I ◽

Spine Density ◽

Shock Response ◽

Anti Inflammatory

(1) Background: Amyotrophic lateral sclerosis (ALS) is a multifactorial non-cell autonomous disease where activation of microglia and astrocytes largely contributes to motor neurons death. Heat shock proteins have been demonstrated to promote neuronal survival and exert a strong anti-inflammatory action in glia. Having previously shown that the pharmacological increase of the histamine content in the central nervous system (CNS) of SOD1-G93A mice decreases neuroinflammation, reduces motor neuron death, and increases mice life span, here we examined whether this effect could be mediated by an enhancement of the heat shock response. (2) Methods: Heat shock protein expression was analyzed in vitro and in vivo. Histamine was provided to primary microglia and NSC-34 motor neurons expressing the SOD1-G93A mutation. The brain permeable histamine precursor histidine was chronically administered to symptomatic SOD1-G93A mice. Spine density was measured by Golgi-staining in motor cortex of histidine-treated SOD1-G93A mice. (3) Results: We demonstrate that histamine activates the heat shock response in cultured SOD1-G93A microglia and motor neurons. In SOD1-G93A mice, histidine augments the protein content of GRP78 and Hsp70 in spinal cord and cortex, where the treatment also rescues type I motor neuron dendritic spine loss. (4) Conclusion: Besides the established histaminergic neuroprotective and anti-inflammatory effects, the induction of the heat shock response in the SOD1-G93A model by histamine confirms the importance of this pathway in the search for successful therapeutic solutions to treat ALS.

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