Defective Repair of Uracil Causes Telomere Defects in Mouse Hematopoietic Cells

Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity.     

Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes

Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. We improved a previously described gas chromatography-mass spectrometry (GC-MS) assay for uracil in DNA and validated the assay by analyzing the DNA-uracil content of normal, primary human lymphocytes that were cultured in 0-3000 nM folic acid. In addition, the effects of nucleoside mixtures T or TdCA (T, thymidine; A, adenosine; dC, deoxycytidine) were investigated. Over 4 consecutive days, the inter- and intraassay coefficients of variation (CVs) were 2.3-3.9 and 0.6-2.2%. Mean recovery was 99.4%. Oligonucleotides containing 100 pg of uracil yielded a mean uracil measurement of 110.1 pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12 nM, and minima at 0 and 3000 nM folate. Extremely folate-deficient cells may incorporate less uracil because DNA synthesis is reduced. A wide response to folate deficiency was seen in cells from different donors, suggesting that genetic background plays a critical role in individual susceptibility to DNA damage and cancer risk. Unexpectedly, TdCA supplementation caused increased DNA-uracil (vs 3000 nM folate for 10 days, P > 0.05), probably due to the conversion of deoxycytidine to deoxyuridine by cytidine deaminase, leading to elevated dUMP/dTMP ratios. This improved uracil assay could serve as a useful tool in the study of the mechanism of uracil misincorporation into DNA. The assay requires 3 microg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans.     

Ginsenoside Rg1 and Acori Graminei Rhizoma Attenuates Neuron Cell Apoptosis by Promoting the Expression of miR-873-5p in Alzheimer’s Disease

Alzheimer’s disease (AD) severely threatens human health in their old age, however the potential etiology underlying it is still unclear. Both Ginsenoside Rg1 (GRg1) and Acori graminei Rhizoma (AGR) are the traditional Chinese herbal drug, while their potential role in AD remains need further identification. Both SAMP1 and SAMP8 mice were employed as the control and AD mice. Morris water maze method was used to detect the cognitive function of the mice, TUNEL assay was performed to determine cell apoptosis. Real-time PCR and western blot were carried out to measure gene expression. The relationship between miR-873-5p and HMOX1 was determined using luciferase reporter assay. Comparing with SAMP1, the cognitive function was impaired and cell apoptosis was increased in SAMP8 mice. GRg1?+?AGR treatment significantly attenuated the symptom of AD. The expression of miR-873-5p was decreased, while HMOX1 was increased in SAMP8 mice. GRg1?+?AGR treatment significantly promoted the expression of miR-873-5p, but decreased HMOX1. MiR-873-5p targets HMOX1 to regulate its expression. Aβ1–42 stimulation decreased the expression of miR-873-5p, but increased HMOX1 in PC12 cells. GRg1?+?AGR treatment reversed the effect of Aβ1–42, while miR-873-5p inhibitor abolished the effect of GRg1?+?AGR. In vivo experiments confirmed the protect role of GRg1?+?AGR in AD. GRg1?+?AGR suppressed neuron cell apoptosis by regulating the expression of miR-873-5p in AD.     

Oxidative damage to RNA and expression patterns of MTH1 in the hippocampi of senescence-accelerated SAMP8 mice and Alzheimer's disease patients

Mammalian MTH1 protein, a MutT-related protein, catalyzes the hydrolysis of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxoGTP) to monophosphate, thereby preventing incorporation of 8-oxo-7,8-dihydroguanine (8-oxoguanine) into RNA. In this study, we applied immunohistochemistry to follow the expression of MTH1 and the amount of 8-oxoguanine in RNA during aging. There were increased amounts of 8-oxoguanine in RNA in the CAl and CA3 subregions of hippocampi of 8- and 12-month-old SAMP8 mice, which exhibited early aging syndromes and declining learning and memory abilities compared to those of age-matched control SAMR1 mice. The expression levels of MTH1 in the hippocampi of 8- and 12-month-old SAMP8 mice were significantly lower than those of control mice. Therefore, in this mouse model, age-related accumulation of 8-oxoguanine in RNA is correlated with decreased expression of MTH1. Increased amounts of 8-oxoguanine in the RNA, and decreased expression of MTH1 were also observed in the hippocampi of patients suffering from Alzheimer’s disease. These results suggest that MTH1 deficiency might be a causative factor for aging and age-related disorders.     

Suppression of uracil-DNA glycosylase induces neuronal apoptosis

A chronic imbalance in DNA precursors, caused by one-carbon metabolism impairment, can result in a deficiency of DNA repair and increased DNA damage. Although indirect evidence suggests that DNA damage plays a role in neuronal apoptosis and in the pathogenesis of neurodegenerative disorders, the underlying mechanisms are poorly understood. In particular, very little is known about the role of base excision repair of misincorporated uracil in neuronal survival. To test the hypothesis that repair of DNA damage associated with uracil misincorporation is critical for neuronal survival, we employed an antisense (AS) oligonucleotide directed against uracil-DNA glycosylase encoded by the UNG gene to deplete UNG in cultured rat hippocampal neurons. AS, but not a scrambled control oligonucleotide, induced apoptosis, which was associated with DNA damage analyzed by comet assay and up-regulation of p53. UNG mRNA and protein levels were decreased within 30 min and were undetectable within 6-9 h of exposure to the UNG AS oligonucleotide. Whereas UNG expression is significantly higher in proliferating as compared with nonproliferating cells, such as neurons, the levels of UNG mRNA were increased in brains of cystathionine beta-synthase knockout mice, a model for hyperhomocysteinemia, suggesting that one-carbon metabolism impairment and uracil misincorporation can induce the up-regulation of UNG expression.     

Differential expression of the liver proteome in senescence accelerated mice

The senescence-accelerated mouse (SAM) is a useful animal model to study aging or age-associated disorders due to its inherited aging phenotype. To investigate proteins involved in the aging process in liver, we compared the young (4- or 20-week old) and the aged group (50-week-old) of SAMP8 (short life span) and SAMR1 (control) mice, and identified 85 differentially expressed distinct proteins comprising antioxidation, glucose/amino acid metabolism, signal transduction and cell cycle systems using proteomics tools. For the antioxidation system, the aged SAMP8 mice showed a large increase in glutathione peroxidase and decreases in glutathione-S-transferase and peroxiredoxin, ranging from 2.5- to 5-fold, suggesting lower detoxification potentials for oxidants in the aged SAMP8 liver. Similarly, levels of key glycolytic enzymes decreased greatly in the aged SAMP8 compared to SAMR1, indicating a disturbance in glucose homeostasis that may be closely related to the typical deficits in learning and memory of the aged SAMP8. Protein profiles of amino acid metabolic enzymes suggest that accumulation of glutamine and glutamate in tissues of the aged SAMP8 may be due to hyperexpression of ornithine aminotransferase and/or glutamate dehydrogenase. Decreases in levels of proteins involved in signal transduction/apoptosis (e.g., cathepsin B) in the aged SAMP8 may support the previously proposed negative relationship between apoptosis and aging. However, the changes described above were not markedly seen in the young group of both strains. For cell cycle systems, levels of selenium binding protein increased about four-fold with age in SAMP8. Yet, almost no change occurred in either the young or the aged SAMR1, which may explain problems associated with cell proliferation and tissue regeneration in the aged SAMP8. In conclusion, composite profiles of key proteins involved in age-related processes enable assessment of accelerated senescence and the appearance of senescence-related pathologies in the aged SAMP8.     

Crystal structure of family 5 uracil-DNA glycosylase bound to DNA

Uracil-DNA glycosylase (UDG) removes uracil generated by the deamination of cytosine or misincorporation of deoxyuridine monophosphate. Within the UDG superfamily, a fifth UDG family lacks a polar residue in the active-site motif, which mediates the hydrolysis of the glycosidic bond by activation of a water molecule in UDG families 1-4. We have determined the crystal structure of a novel family 5 UDG from Thermus thermophilus HB8 complexed with DNA containing an abasic site. The active-site structure suggests this enzyme uses both steric force and water activation for its excision reaction. A conserved asparagine residue acts as a ligand to the catalytic water molecule. The structure also implies that another water molecule acts as a barrier during substrate recognition. Based on no significant open-closed conformational change upon binding to DNA, we propose a "slide-in" mechanism for initial damage recognition.     

Beta-amyloid precursor polypeptide in SAMP8 mice affects learning and memory

Senescence accelerated (SAMP8 [P8]) mice develop age-related deficits in memory and learning. We show that increased expression of amyloid precursor protein (APP) and its mRNA in the hippocampus are also age-related. Immunocytochemical data suggest that a critical amount of APP expression may be needed to generate amyloid (Aβ) protein plaques in the hippocampus. Deficits in acquisition and retention test performance were alleviated by administration of antibody to Aβ protein into the cerebral ventricles. This reversal of cognitive deficits provides a link between increased expression of both APP and Aβ protein and learning and memory loss in these mice.     

MeCP2 prevents age‐associated cognitive decline via restoring synaptic plasticity in a senescence‐accelerated mouse model

Age‐related cognitive decline in neurodegenerative diseases, such as Alzheimer''s disease (AD), is associated with the deficits of synaptic plasticity. Therefore, exploring promising targets to enhance synaptic plasticity in neurodegenerative disorders is crucial. It has been demonstrated that methyl‐CpG binding protein 2 (MeCP2) plays a vital role in neuronal development and MeCP2 malfunction causes various neurodevelopmental disorders. However, the role of MeCP2 in neurodegenerative diseases has been less reported. In the study, we found that MeCP2 expression in the hippocampus was reduced in the hippocampus of senescence‐accelerated mice P8 (SAMP8) mice. Overexpression of hippocampal MeCP2 could elevate synaptic plasticity and cognitive function in SAMP8 mice, while knockdown of MeCP2 impaired synaptic plasticity and cognitive function in senescence accelerated‐resistant 1 (SAMR1) mice. MeCP2‐mediated regulation of synaptic plasticity may be associated with CREB1 pathway. These results suggest that MeCP2 plays a vital role in age‐related cognitive decline by regulating synaptic plasticity and indicate that MeCP2 may be promising targets for the treatment of age‐related cognitive decline in neurodegenerative diseases.     

Testosterone deficiency accelerates neuronal and vascular aging of SAMP8 mice: protective role of eNOS and SIRT1

Oxidative stress and atherosclerosis-related vascular disorders are risk factors for cognitive decline with aging. In a small clinical study in men, testosterone improved cognitive function; however, it is unknown how testosterone ameliorates the pathogenesis of cognitive decline with aging. Here, we investigated whether the cognitive decline in senescence-accelerated mouse prone 8 (SAMP8), which exhibits cognitive impairment and hypogonadism, could be reversed by testosterone, and the mechanism by which testosterone inhibits cognitive decline. We found that treatment with testosterone ameliorated cognitive function and inhibited senescence of hippocampal vascular endothelial cells of SAMP8. Notably, SAMP8 showed enhancement of oxidative stress in the hippocampus. We observed that an NAD(+)-dependent deacetylase, SIRT1, played an important role in the protective effect of testosterone against oxidative stress-induced endothelial senescence. Testosterone increased eNOS activity and subsequently induced SIRT1 expression. SIRT1 inhibited endothelial senescence via up-regulation of eNOS. Finally, we showed, using co-culture system, that senescent endothelial cells promoted neuronal senescence through humoral factors. Our results suggest a critical role of testosterone and SIRT1 in the prevention of vascular and neuronal aging.     

Uridine enhances expression of the fragile X chromosome in human lymphocytes

Summary We have studied the effect of uridine on the expression of fragile X (fra[X]) in lymphocyte cultures established in the folate and thymidine deficient medium TC199. The results indicate that uridine enhances the expression of fra(X) and gives a higher mitotic rate. The excess of uridine during DNA synthesis might further promote the previously suggested cycle of misincorporation and removal of deoxyuridine monophosphate when the pool of deoxythymidine triphosphate is continuously depleted.     

Butyric acid alleviated chronic intermittent hypoxia-induced lipid formation and inflammation through up-regulating HuR expression and inactivating AMPK pathways

To investigate whether butyric acid could alleviate chronic intermittent hypoxia (CIH)-induced lipid formation in human preadipocytes-subcutaneous (HPA-s) through accumulation of human antigen R (HuR) and inactivation of AMP-activated protein kinase (AMPK) pathway, HPA-s were obtained and divided into three groups: Control group: cells were cultured under normal conditions; CIH group: cells were cultured in a three-gas incubator (10% O₂); Butyric acid group: 10 mmol/l butyric acid added into cell culture medium. HuR-siRNA was futher transfected into CIH group for verification the function of HuR. Oil Red O was implemented for observation of lipid droplets within cells. Cell Counting Kit-8 (CCK8) assay was used for detecting cell viability. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) assay as well as flow cytometry analysis was employed for determining cell apoptosis. Western blotting was used for measurement of protein expression levels. RT-qPCR analysis was used for detecting mRNA expression. CIH treatment increased adipocytes proliferation, while butyric acid inhibited cell proliferation and promoted cell apoptosis. The treatment of butyric acid in CIH group down-regulated expression of inflammatory factors and increased cell apoptotic rate. Butyric acid treatment increased HuR expression in both cytoplasm and nucleus and decreased the level of p-AMPK and p-ACC, while transfection of AMPK activator or HuR-siRNA would down-regulate HuR expression. Moreover, butyric acid alleviated CIH-induced cell proliferation, lipid formation and inflammatory status and promoted cell apoptosis through regulating related genes including p21, PPARγ, C/EBPa, IL-1β, IL-6, TLR4, caspase-8 and caspase-3. In conclusion, butyric acid could alleviate CIH-induced inflammation, cell proliferation and lipid formation through accumulation of HuR and inactivation of AMPK pathway.     

Tea catechin ingestion combined with habitual exercise suppresses the aging-associated decline in physical performance in senescence-accelerated mice

Catechins, which are abundant in green tea, possess a variety of biologic actions, and their clinical application has been extensively investigated. In this study, we examined the effects of tea catechins and regular exercise on the aging-associated decline in physical performance in senescence-accelerated prone mice (SAMP1) and age-matched senescence-accelerated resistant mice (SAMR1). The endurance capacity of SAMR1 mice, measured as the running time to exhaustion, tended to increase over the 8-wk experimental period, whereas that of SAMP1 mice decreased by 17%. On the other hand, the endurance capacity of SAMP1 mice fed 0.35% (wt/wt) catechins remained at the initial level and was significantly higher than that of SAMP1 mice not fed catechins. In SAMP1 mice fed catechins and given exercise, oxygen consumption was significantly increased, and there was an increase in skeletal muscle fatty acid beta-oxidation. The mRNA levels of mitochondria-related molecules, such as peroxisome proliferator-activated receptor-gamma coactivator-1, cytochrome c oxidase-II, III, and IV in skeletal muscle were also higher in SAMP1 mice given both catechins and exercise. Moreover, oxidative stress measured as thiobarbituric reactive substances was lower in SAMP1 groups fed catechins than in the SAMP1 control group. These results suggest that long-term intake of catechins, together with habitual exercise, is beneficial for suppressing the aging-related decline in physical performance and energy metabolism and that these effects are due, at least in part, to improved mitochondrial function in skeletal muscle.     

The Hippocampal Proteomic Analysis of Senescence-Accelerated Mouse: Implications of Uchl3 and Mitofilin in Cognitive Disorder and Mitochondria Dysfunction in SAMP8

Senescence-accelerated mouse prone 8 (SAMP8) is considered as a useful animal model for age-related learning and memory impairments. Hippocampus, a critical brain region associated with cognitive decline during normal aging and various neurodegenerative diseases, appeared a series of abnormalities in SAMP8. To investigate the molecular mechanisms underlying age-related cognitive disorders, we used 2-DE coupled with MALDI TOF/TOF MS to analyze the differential protein expression of the hippocampus of SAMP8 at 6-month-old compared with the age-matched SAM/resistant 1 (SAMR1) which shows normal aging process. Two proteins were found to be markedly changed in SAMP8 as compared to SAMR1: ubiquitin carboxyl-terminal hydrolase L3 (Uchl3), implicating in cytosolic proteolysis of oxidatively damaged proteins, was down-regulated while mitofilin, a vital protein for normal mitochondria function, exhibited four isoforms with a consistent basic shift of isoelectric point among the soluble hippocampal proteins in SAMP8 compared with SAMR1. The alterations were confirmed by Western blotting analysis. The analysis of their expression changes may shed light on the mechanisms of learning and memory deficits and mitochondrial dysfunction as observed in SAMP8.     

Folic Acid Supplementation Promotes Mammary Tumor Progression in a Rat Model

Folic acid supplementation may prevent the development of cancer in normal tissues but may promote the progression of established (pre)neoplastic lesions. However, whether or not folic acid supplementation can promote the progression of established (pre)neoplastic mammary lesions is unknown. This is a critically important issue because breast cancer patients and survivors in North America are likely exposed to high levels of folic acid owing to folic acid fortification and widespread supplemental use after cancer diagnosis. We investigated whether folic acid supplementation can promote the progression of established mammary tumors. Female Sprague-Dawley rats were placed on a control diet and mammary tumors were initiated with 7,12-dimethylbenza[a]anthracene at puberty. When the sentinel tumor reached a predefined size, rats were randomized to receive a diet containing the control, 2.5x, 4x, or 5x supplemental levels of folic acid for up to 12 weeks. The sentinel mammary tumor growth was monitored weekly. At necropsy, the sentinel and all other mammary tumors were analyzed histologically. The effect of folic acid supplementation on the expression of proteins involved in proliferation, apoptosis, and mammary tumorigenesis was determined in representative sentinel adenocarcinomas. Although no clear dose-response relationship was observed, folic acid supplementation significantly promoted the progression of the sentinel mammary tumors and was associated with significantly higher sentinel mammary tumor weight and volume compared with the control diet. Furthermore, folic acid supplementation was associated with significantly higher weight and volume of all mammary tumors. The most significant and consistent mammary tumor-promoting effect was observed with the 2.5x supplemental level of folic acid. Folic acid supplementation was also associated with an increased expression of BAX, PARP, and HER2. Our data suggest that folic acid supplementation may promote the progression of established mammary tumors. The potential tumor-promoting effect of folic acid supplementation in breast cancer patients and survivors needs further clarification.     

Proteomic analysis of specific brain proteins in aged SAMP8 mice treated with alpha-lipoic acid: implications for aging and age-related neurodegenerative disorders

Free radical-mediated damage to neuronal membrane components has been implicated in the etiology of Alzheimer's disease (AD) and aging. The senescence accelerated prone mouse strain 8 (SAMP8) exhibits age-related deterioration in memory and learning along with increased oxidative markers. Therefore, SAMP8 is a suitable model to study brain aging and, since aging is the major risk factor for AD and SAMP8 exhibits many of the biochemical findings of AD, perhaps as a model for and the early phase of AD. Our previous studies reported higher oxidative stress markers in brains of 12-month-old SAMP8 mice when compared to that of 4-month-old SAMP8 mice. Further, we have previously shown that injecting the mice with alpha-lipoic acid (LA) reversed brain lipid peroxidation, protein oxidation, as well as the learning and memory impairments in SAMP8 mice. Recently, we reported the use of proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. In order to understand how LA reverses the learning and memory deficits of aged SAMP8 mice, in the current study, we used proteomics to compare the expression levels and specific carbonyl levels of proteins in brains from 12-month-old SAMP8 mice treated or not treated with LA. We found that the expressions of the three brain proteins (neurofilament triplet L protein, alpha-enolase, and ubiquitous mitochondrial creatine kinase) were increased significantly and that the specific carbonyl levels of the three brain proteins (lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and alpha-enolase) were significantly decreased in the aged SAMP8 mice treated with LA. These findings suggest that the improved learning and memory observed in LA-injected SAMP8 mice may be related to the restoration of the normal condition of specific proteins in aged SAMP8 mouse brain. Moreover, our current study implicates neurofilament triplet L protein, alpha-enolase, ubiquitous mitochondrial creatine kinase, lactate dehydrogenase B, and dihydropyrimidinase-like protein 2 in process associated with learning and memory of SAMP8 mice.     

Curcumin prevents mitochondrial dysfunction in the brain of the senescence-accelerated mouse-prone 8

The aging brain suffers mitochondrial dysfunction and a reduced availability of energy in the form of ATP, which in turn may cause or promote the decline in cognitive, sensory, and motor function observed with advancing age. There is a need for animal models that display some of the pathological features of human brain aging in order to study their prevention by e.g. dietary factors. We thus investigated the suitability of the fast-aging senescence-accelerated mouse-prone 8 (SAMP8) strain and its normally aging control senescence-accelerated mouse-resistant 1 (SAMR1) as a model for the age-dependent changes in mitochondrial function in the brain. To this end, 2-months old male SAMR1 (n = 10) and SAMP8 mice (n = 7) were fed a Western type diet (control groups) for 5 months and one group of SAMP8 mice (n = 6) was fed an identical diet fortified with 500 mg curcumin per kg. Dissociated brain cells and brain tissue homogenates were analyzed for malondialdehyde, heme oxygenase-1 mRNA, mitochondrial membrane potential (MMP), ATP concentrations, protein levels of mitochondrial marker proteins for mitochondrial membranes (TIMM, TOMM), the mitochondrial permeability transition pore (ANT1, VDAC1, TSPO), respiration complexes, and fission and fusion (Fis, Opa1, Mfn1, Drp1). Dissociated brain cells isolated from SAMP8 mice showed significantly reduced MMP and ATP levels, probably due to significantly diminished complex V protein expression, and increased expression of TSPO. Fission and fusion marker proteins indicate enhanced mitochondrial fission in brains of SAMP8 mice. Treatment of SAMP8 mice with curcumin improved MMP and ATP and restored mitochondrial fusion, probably by up-regulating nuclear factor PGC1α protein expression. In conclusion, SAMP8 compared to SAMR1 mice are a suitable model to study age-dependent changes in mitochondrial function and curcumin emerges as a promising nutraceutical for the prevention of neurodegenerative diseases that are accompanied or caused by mitochondrial dysfunction.     

Folate deficiency inhibits the proliferation of primary human CD8+ T lymphocytes in vitro

Folate is required for one-carbon transfer reactions and the formation of purines and pyrimidines for DNA and RNA synthesis. Deficiency of folate can lead to many clinical abnormalities, including macrocytic anemia, cardiovascular diseases, birth defects, and carcinogenesis. The nucleotide imbalance due to folate deficiency causes cell cycle arrest in the S phase and uracil misincorporation into DNA, which may result in DNA double-strand breaks during repair. The role of folate in the immune system has not been fully characterized. We cultured PHA-activated human T lymphocytes in varying concentrations of folate, and measured proliferation, cell cycle, apoptosis, uracil misincorporation, and proportions of Th cells (CD4(+)) and cytotoxic T (CD8(+)) cells. Folate deficiency reduced proliferation of T lymphocytes, induced cell cycle arrest in the S phase, induced apoptosis, and increased the level of uracil in DNA. Folate deficiency also increased the CD4(+) to CD8(+) ratio due to a marked reduction of CD8(+) cell proliferation. Folate or nucleoside repletion of folate-deficient cells rapidly restored T lymphocyte proliferation and normal cell cycle, reduced the DNA uracil content, and lowered the CD4(+) to CD8(+) ratio. These data suggest that folate status may affect the immune system by reducing the capacity of CD8(+) cells to proliferate in response to activation.