The ATM-BID pathway regulates quiescence and survival of haematopoietic stem cells

BID, a BH3-only BCL2 family member, functions in apoptosis as well as the DNA-damage response. Our previous data demonstrated that BID is an ATM effector acting to induce cell-cycle arrest and inhibition of apoptosis following DNA damage. Here we show that ATM-mediated BID phosphorylation plays an unexpected role in maintaining the quiescence of haematopoietic stem cells (HSCs). Loss of BID phosphorylation leads to escape from quiescence of HSCs, resulting in exhaustion of the HSC pool and a marked reduction of HSC repopulating potential in vivo. We also demonstrate that BID phosphorylation plays a role in protecting HSCs from irradiation, and that regulating both quiescence and survival of HSCs depends on BID's ability to regulate oxidative stress. Moreover, loss of BID phosphorylation, ATM knockout or exposing mice to irradiation leads to an increase in mitochondrial BID, which correlates with an increase in mitochondrial oxidative stress. These results show that the ATM-BID pathway serves as a critical checkpoint for coupling HSC homeostasis and the DNA-damage stress response to enable long-term regenerative capacity.     

Effect of egg‐phosphatidylcholine on the chilling sensitivity and lipid phase transition of Asian elephant (Elephas maximus) spermatozoa

This study was conducted in an effort to improve our understanding of the response of Asian elephant (Elephas maximus, Em) spermatozoa to chilling. Semen was collected from two elephant bulls by means of the manual rectal stimulation method. Five out of seven semen collections were deemed to be suitable for use based on motility (ranging from 20% to 60%) and membrane integrity. We evaluated the chilling sensitivity by incubating the sperm with a fluorescent dye (5‐carboxyfluorescein diacetate (cFDA)) at 16°C, 12°C, 4°C, and 22°C (control). Cells with an intact membrane retained the dye and were identified as viable. The membrane lipid phase transition (LPT) temperature curve was determined with a Fourier transform infrared (FTIR) spectrometer connected to an FTIR microscope. The LPT center, T_m, was determined by statistical analysis. The LPT and T_m were also assessed in fresh spermatozoa and spermatozoa incubated with egg yolk or egg‐phosphatidylcholine (EPC) liposomes at 16°C, 12°C, 4°C, and 26°C (control). The results show that the membrane integrity of spermatozoa incubated at 16°C, 12°C, and 4°C decreased by 39%, 62%, and 67%, respectively, compared to the control. The LPT temperatures were between room temperature (26°C) and 10°C, with T_m at 14–16°C. The T_m for sperm incubated with liposomes or egg‐yolk extender was below the measured range (2°C). Chilling sensitivity was found at a wide range of temperatures and transition temperatures, suggesting the presence of a wide variety of fatty acids (FAs) in the membrane with a high ratio of saturated‐to‐polyunsaturated FAs. Here we show that the protection afforded by the presence of egg yolk or liposomes in the extender is accomplished by shifting the T_m to below the 4°C point at which chilled semen is maintained for transport, or the point at which fast freezing begins to minimize cellular damage. Zoo Biol 0:1–13, 2005. © 2005 Wiley‐Liss, Inc.     

Prolonged, granulocyte–macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

Introduction

A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (T_H17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect T_H17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.

Methods

IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3'-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.

Results

T_H17-expressing CD4⁺ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASF_IL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASF_IL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASF_IL-17/TNF-mediated neutrophil rescue.

Conclusion

The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. T_H17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.     

Antagonism of the estradiol-mediated repression of microsomal 3β-hydroxysteroid dehydrogenase activity in rat liver by antiestrogenic substances

5α-Dihydrotestosterone, 17-hydroxyprogesterone caproate, 2-methoxyestrone and a number of nonsteroidal antiestrogens (clomiphene citrate, nafoxidine hydrochloride, tamoxifen, MER-25) were tested for their ability to block estradiol-mediated repression of the androgen-dependent 3β-hydroxy-steroid dehydrogenase activity of male rat liver. With the exception of 5α-dihydrotestosterone, which induced activity in females, none of these substances affected 3β-hydroxy-steroid dehydrogenase activity when administered alone to otherwise untreated male and female rats. Tamoxifen (100 or 500 μg/day) was the only substance which prevented a decrease in enzyme activity when given simultaneously with estradiol (5 μg/day). The estradiol-mediated decrease in activity was not antagonized by a 100-fold higher dose of androgen (5α-dihydrotestosterone, 0.5 mg/day), demonstrating the potent antiandrogenic effect of estradiol on this hepatic androgen-dependent enzyme activity.     

Functional analysis of the ligand binding site of EGF-receptor utilizing chimeric chicken/human receptor molecules.

The epidermal growth factor (EGF)-receptor is composed of an extracellular ligand-binding region connected by a single transmembrane region to the cytoplasmic kinase domain. In spite of its importance for understanding signal transduction, the ligand-binding domain of the EGF-receptor is not yet defined. We describe the identification of a major ligand-binding domain of the EGF-receptor by utilizing chimeras between the human EGF-receptor and the chicken EGF-receptor. This approach is based on the fact that murine EGF binds to the chicken EGF-receptor with 100-fold lower affinity as compared to the human EGF-receptor. Hence, the substitution of various domains of the chicken EGF-receptor by domains of the human EGF-receptor may restore the higher binding affinity towards EGF, characteristic of the human receptor. We show that chimeric chicken/human EGF-receptor, which contains domain III of the extracellular region of the human receptor, behaves like the human EGF-receptor with respect to EGF binding affinity and biological responsiveness. However, a chimeric chicken/human EGF-receptor containing domains I and II of the human receptor behaves like the chicken rather than the human EGF-receptor. Moreover, two different monoclonal antibodies which compete for the binding of EGF to EGF-receptor recognize specifically domain III of the human EGF-receptor. It is concluded that domain III which is flanked by the two cysteine-rich domains is a major ligand-binding domain of the EGF-receptor.     

Activation of c-erbB in avian leukosis virus-induced erythroblastosis leads to the expression of a truncated EGF receptor kinase.

Chicken erythroblastosis caused by avian leukosis virus (ALV) is thought to be mediated by activation of the c-erbB/EGF receptor oncogene by a promoter-insertion mechanism. Here we study the proteins expressed by two ALV-induced leukemias and compare them with the avian EGF receptor and with the oncogene product of avian erythroblastosis virus (v-erbB) which was shown to be a truncated EGF receptor. It appears that the two leukemias express truncated EGF receptors of slightly different sizes with intrinsic tyrosine kinase activity. Hence, acute and chronic retroviruses utilize a common pathway for transformation. Moreover, the proteins expressed in the leukemias are similar to the avian EGF receptor with respect to their phosphopeptide maps, suggesting that they do not carry the C-terminal deletion characteristic of v-erbB.