While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the "anti-aging" protein, ...α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age.
Protein tyrosine kinases play a major role in promoting cell growth, and their activity in solid tumors is well established. Inhibitors of protein tyrosine kinases are now in advanced clinical trials ...for the treatment of breast and brain cancers. Because Src-related PTK have been shown to be activated in leukemic cell lines, we studied their activation in human myeloid leukemia. Blasts from the majority of patients with acute leukemia showed constitutive activity of the Src kinase Lyn. In contrast, no patient samples showed constitutive activation of Jak2. Genetic and pharmacologic targeting of Lyn was used to determine its contribution to leukemic cell growth. Antisense Lyn oligonucleotide treatment resulted in the inhibition of tritiated thymidine incorporation following GM-CSF stimulation of the factor-dependent line MO7e. The Src kinase inhibitor PD166285 inhibited the growth of human leukemic cell lines and leukemic blasts. When combined with doxorubicin, an additive effect on the inhibition of leukemic cell growth occurred. These studies demonstrate the importance of Src kinases in promoting leukemic cell growth and suggests that further development of agents which target Src kinases and their inclusion in multidrug regimens are warranted for novel therapies of myeloid leukemia.
Nucleotide excision repair (NER) acts on a variety of DNA lesions, including damage induced by many chemotherapeutic drugs. Cancer therapy with such drugs might be improved by reducing the NER ...capacity of tumors. It is not known, however to what extent any individual NER protein is rate-limiting for any step of the repair reaction. We studied sensitivity to UV radiation and repair of DNA damage with regard to XPA, one of the core factors in the NER incision complex. About 150,000–200,000 molecules of XPA protein are present in NER proficient human cell lines, and no XPA protein in the XP-A cell line XP12RO. Transfected XP12RO cell lines expressing 50,000 or more XPA molecules/cell showed UV resistance similar to normal cells. Suppression of XPA protein to ∼10,000
molecules/cell in a Tet-regulatable system modestly but significantly increased sensitivity to UV irradiation. No removal of cyclobutane pyrimidine dimers was detected in the SV40 immortalized cell lines tested. Repair proficient WI38-VA fibroblasts and transfected XP-A cells expressing 150,000 molecules of XPA/cell removed (6–4) photoproducts from the genome with a half-life of 1
h. Cells in which XPA protein was reduced to about 10,000
molecules/cell removed (6–4) photoproducts more slowly, with a half-life of 3
h. A reduced rate of repair of (6–4) photoproducts thus results in increased cellular sensitivity towards UV irradiation. These data indicate that XPA levels must be reduced to <10% of that present in a normal cell to render XPA a limiting factor for NER and consequent cellular sensitivity. To inhibit NER, it may be more effective to interfere with XPA protein function, rather than reducing XPA protein levels.
Engagement of the Granulocyte-Colony-Stimulating Factor (G-CSF) receptor activates non-receptor protein tyrosine kinases Lyn and Jak2. We found that Lyn-deficient DT40 cells that express the G-CSF ...receptor (DT40GR) do not demonstrate G-CSF-induced mitogenic signaling. Lyn associates with and phosphorylates a small set of molecules, including c-Cbl. c-Cbl is an adaptor involved in cell growth and cytoskeletal reorganization, predominantly in hematopoietic cells. Using yeast two-hybrid analysis, we found that c-Cbl directly couples Lyn to PI 3-kinase. We also found that expression of the c-CblY731F mutant, which uncouples PI 3-kinase, resulted in the inhibition of G-CSF-induced proliferative signaling in DT40GR cells. As a complementary strategy, we sought to analyse the effects of c-Cbl deficiency in DT40GR cells. We isolated, cloned and sequenced the full-length cDNA for chicken c-Cbl and constructed antisense vectors. Antisense inhibition of c-Cbl expression in DT40GR cells led to enhanced Jak-STAT activation following G-CSF stimulation. Yet, this enhancement of Jak-STAT activation was associated with decreased G-CSF-induced PI 3-kinase activity and DNA synthesis. PI 3-kinase activity correlated with DNA synthesis and physiological levels of c-Cbl. Together, these data suggest that physiologic level of c-Cbl provides a growth stimulatory pathway for G-CSF and that enhanced Jak-STAT activation is not sufficient for G-CSF-induced growth.
Granulocyte colony-stimulating factor (G-CSF) is the major hematopoietic factor which controls the production and differentiation of granulocytes. The G-CSF receptor (G-CSFR) belongs to the ...superfamily of the cytokine receptors, which transduce signals via the activation of cytosolic protein tyrosine kinases (PTK). To determine the role of specific PTK in G-CSF signaling we expressed the human G-CSFR in cell lines derived from DT40 B cells, which lack either the Src-related Lyn or Syk. Wild-type (wt) and syk-deficient cells underwent increased DNA synthesis in response to G-CSF; lyn-deficient cells did not. The purpose of these studies is to identify Lyn's downstream effectors in mediating DNA synthesis. While G-CSF stimulated Ras activity in all cell lines, G-CSF failed to induce the tyrosine phosphorylation of Shc in lyn-deficient cells. G-CSF induced a statistically significant activation of Erk1/Erk2 Kinase or p90Rsk only in the wt cells. G-CSF induced the tyrosine phosphorylation of Cbl and increased activity of PI 3-kinase in wild-type and syk-deficient, but non in lyn-deficient, cells. Inhibition of Shc by over-expression of its SH2 or PTB domains or PI 3-kinase by either treatment with wortmannin or expression of the CblY731F mutant decreased G-CSF-induced DNA synthesis. Thus, the Lyn, Cbl-PI 3-kinase, and Shc/non-Ras-dependent pathways correlate with the ability of cells to respond to G-CSF with increased DNA synthesis.
The retroviral oncogene v-Cbl causes pre-B cell lymphomas and myeloid leukemias in mice, and its
Drosophila homologue is oncogenic, causing enhanced receptor tyrosine kinase signaling. The human Cbl ...gene resides at 11q23. The aim of this study is to determine the effect of oncogenic Cbl on growth-regulating responses.
The oncogenic mutant of Cbl (CblΔ1-357) was transfected into factor-dependent 32Dcl3 myeloid cells. Consequently, cell survival and differentiation were measured. Lyn, Syk, MAP kinase, and phosphatidylinositol 3′(PI3′)-kinase activities, protein phosphorylation, Bcl-2 promoter activity, ubiquitination, and levels of Bcl-2, Bax, Bad, and Bcl-x
L were determined. In addition, the effect of v-Cbl on TF-1 cell survival upon granulocyte-macrophage colony-stimulating factor withdrawal was studied.
32Dcl3 and TF-1 cells expressing v-Cbl showed resistance to apoptosis upon growth factor withdrawal, and 32Dcl3 cells completely failed to respond to granulocyte colony-stimulating factor's induction of differentiation. Basal activities of Lyn, Syk, and PI3′-kinase were elevated in the v-Cbl line. There was neither enhanced tyrosine phosphorylation of cellular protein content, Cbl, or Jak2, nor serine phosphorylation of MAP kinase or Akt. After factor withdrawal, the level of Bcl-2 was greater in v-Cbl cells than in control cells.
Neither increased Bcl-2 promoter activity nor decreased ubiquitination of Bcl-2 could account for increased Bcl-2 levels. v-Cbl–expressing 32Dcl3 cells were resistant to differentiation. v-Cbl suppresses apoptosis and differentiation, possibly through enhancement of Lyn, Syk, and PI3′-kinase activities and Bcl-2.
The reactions of silylene centres (SC) on the surface of a chemically activated silica (reactive silica, RSi), characteristic of silylenes and carbenes under homogeneous conditions, have been ...explored by IR and luminescence spectroscopy. SC were found to insert into the HS bond of H
2S and the CS bond of Me
2S with rate constants of 4.5 · 10
−17 cm
3 · molecule
−1 · s
−1 and 8.3 · 10
−18 cm
3 · molecule
−1 · s
−1, respectively. The reaction of SC with ethylene oxide proceeds via ring-opening with a further cyclization to give presumably a 5-membered surface siladioxolane heterocycle. The thermal reaction between SC and ethylene is shown to be very slow and not to yield products of addition or insertion. Ethylene appears to quench both fluorescence and phosphorescence of SC with almost gasokinetical quenching constants, but photoreaction between SC and ethylene was not observed.
With reference to M. N. Slavyatinskaya's (1976) study showing the various meanings of feugo in Homer's texts, depending on the verb usage in the imperfect or aorist tense, 96 instances of its ...occurrence in Herodotus's History are analyzed. It is found that such meanings of feugo as 'flee', 'avoid', & 'run' result not from a particular tense but from the verb's syntagmatic relationships with other lexemes & its functions in such syntagma (eg, predicate, complement, attribute). Z. Dubiel
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