Incorporation of [32P]orthophosphate into inorganic polyphosphates by human granulocytes and other human cell types

Cowling, Randy T.; Birnboim, H.C.

doi:10.1016/s0021-9258(17)36906-5

Cited by 31 publications

(4 citation statements)

References 27 publications

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“…Laser scanning confocal microscopy (A1R; Nikon) was used to follow the changes in polyP concentration, cytosolic [Ca 2+ ] i , mitochondrial Ca 2+ uptake,  m , and mPTP activity. All fluorescence polyP was further confirmed by its characteristic distribution on high resolution polyacrilamide gel, where the polyP sample and standard appear as a characteristic "polyphosphate ladder" (Cowling and Birnboim, 1994). The appearance of this ladder is caused by the fact that both polyP standard and polyP sample from mitochondria are in fact composed of a heterogeneous mixture of polymers of various sizes.…”

Section: Fluorescence Measurementsmentioning

confidence: 83%

Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes

Seidlmayer

Gómez‐García

Blatter

et al. 2012

Journal of General Physiology

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Mitochondrial dysfunction caused by excessive Ca2+ accumulation is a major contributor to cardiac cell and tissue damage during myocardial infarction and ischemia–reperfusion injury (IRI). At the molecular level, mitochondrial dysfunction is induced by Ca2+-dependent opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane, which leads to the dissipation of mitochondrial membrane potential (ΔΨm), disruption of adenosine triphosphate production, and ultimately cell death. Although the role of Ca2+ for induction of mPTP opening is established, the exact molecular mechanism of this process is not understood. The aim of the present study was to test the hypothesis that the adverse effect of mitochondrial Ca2+ accumulation is mediated by its interaction with inorganic polyphosphate (polyP), a polymer of orthophosphates linked by phosphoanhydride bonds. We found that cardiac mitochondria contained significant amounts (280 ± 60 pmol/mg of protein) of short-chain polyP with an average length of 25 orthophosphates. To test the role of polyP for mPTP activity, we investigated kinetics of Ca2+ uptake and release, ΔΨm and Ca2+-induced mPTP opening in polyP-depleted mitochondria. polyP depletion was achieved by mitochondria-targeted expression of a polyP-hydrolyzing enzyme. Depletion of polyP in mitochondria of rabbit ventricular myocytes led to significant inhibition of mPTP opening without affecting mitochondrial Ca2+ concentration by itself. This effect was observed when mitochondrial Ca2+ uptake was stimulated by increasing cytosolic [Ca2+] in permeabilized myocytes mimicking mitochondrial Ca2+ overload observed during IRI. Our findings suggest that inorganic polyP is a previously unrecognized major activator of mPTP. We propose that the adverse effect of polyphosphate might be caused by its ability to form stable complexes with Ca2+ and directly contribute to inner mitochondrial membrane permeabilization.

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Section: Fluorescence Measurementsmentioning

confidence: 83%

Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes

Seidlmayer

Gómez‐García

Blatter

et al. 2012

Journal of General Physiology

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“…Cell lines of human origin used in this study include HeLa, HL-60, and 293T cells (from ATCC; Rockville, MD). Human neutrophils were isolated as described (Cowling and Birnboim 1994). COS-1 cells (ATCC) are of African green monkey origin.…”

Section: Cell Lines Employedmentioning

confidence: 99%

Characterization of a Polyclonal Antibody to Human Thymidylate Synthase Suitable for the Study of Colorectal Cancer Specimens

As¹,

et al. 1999

J Histochem Cytochem.

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SUMMARY Measurement of thymidylate synthase (hTS) using immunohistochemical techniques has been reported in several clinical studies. However, its value as a prognostic indicator is still not clear. To pursue this, we have developed a new rabbit polyclonal antibody, hTS7.4. The antigen was recombinant hTS containing an N-terminal His 6 -tag. Antiserum hTS7.4 detected recombinant hTS by ELISA at a titer of 1:100,000. Western blot analysis of several human cell lines showed a single band of the expected 36-kD molecular size. HeLa cells treated with the TS inhibitor 5-FUdR showed the expected additional band corresponding to the ternary complex of hTS-dFUMP-reduced folate. hTS7.4 detected TS in bacterial, rat, mouse, and monkey cell extracts, and hTS8.3 (a closely related antiserum) immunoprecipitated a 36-kD [ 35 S]-methionine-labeled protein from HeLa extracts. TS was detectable by indirect immunofluorescence in HeLa cells. Proliferating normal human fibroblasts in culture showed staining, but nonproliferating cells did not. Lymphocytes in the germinal center of human tonsil tissue, which are known to be proliferating, stained with hTS7.4 and also with monoclonal antibody TS106. TS may therefore be useful as an immunohistochemical marker of cell proliferation. Normal colon mucosa showed weak staining, whereas some colorectal cancer specimens stained very strongly with hTS7.4. A clinical study of colorectal cancer using this antibody is in progress.

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“…The RT-PCR product from the RNA of human neutrophils had a sequence identical to that of HL-60 cells (not shown). This was gratifying since differentiated HL-60 cells are not identical to neutrophils, the two cells types differing in many details of biochemistry, cell biology, and function (34)(35)(36). Whereas the array of proteins actively synthesized by neutrophils is quite limited, our data from RT-PCR indicate that VAMP-2 is a member of this highly selective group of proteins.…”

Section: Vamp-2mentioning

confidence: 86%

Untitled

et al. 2001

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Degranulation and membrane fusion by neutrophils are essential to host defense. We sought homologues of neuron-specific fusion proteins in human neutrophils and in their precursors, the promyelocytic cell line HL-60. We screened a differentiated HL-60 library and obtained an 848 bp sequence with a 351 bp open reading frame, identical to that published for human VAMP-2 and including 5' and 3' untranslated regions. RNA from HL-60 cells during differentiation into the neutrophil lineage was subjected to Northern blot analysis. which revealed a transcript of approximately 1050 bp at all stages of differentiation. The amount of these transcripts increased approximately threefold during differentiation, a finding confirmed by quantitative RT-PCR. We also detected mRNA for VAMP-2 in human neutrophils and monocytes using RT-PCR. In like fashion, transcripts of syntaxin-4, another fusion protein, were recovered from a neutrophil cDNA library. As with VAMP-2, expression of syntaxin-4 (determined by Northern blots) also increased, but by only 50%, during differentiation of HL-60 cells. These studies demonstrate that neutrophils and their progenitors possess mRNA for the fusion proteins VAMP-2 and syntaxin-4, and that their transcription increases during differentiation, concurrent with the functional maturation of myeloid cells.

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Incorporation of [32P]orthophosphate into inorganic polyphosphates by human granulocytes and other human cell types

Cited by 31 publications

References 27 publications

Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes

Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes

Characterization of a Polyclonal Antibody to Human Thymidylate Synthase Suitable for the Study of Colorectal Cancer Specimens

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