Dyad content is reduced in cardiac myocytes of mice with impaired calmodulin regulation of RyR2.

In cardiac muscle, calmodulin (CaM) regulates the activity of several membrane proteins involved in Ca(2+) homeostasis (CaV1.2; RyR2, SERCA2, PMCA). Three engineered amino acid substitutions in the CaM binding site of the cardiac ryanodine receptor (RyR2) in mice (Ryr2 (ADA/ADA) ) strongly affect cardiac function, with impaired CaM inhibition of RyR2, reduced SR Ca(2+) sequestration, and early cardiac hypertrophy and death (Yamaguchi et al., J Clin Invest 117:1344-1353, 2007). We have examined the ultrastructure and RyR2 immunolocalization in WT and Ryr2 (ADA/ADA) hearts at ~10 days after birth. The myocytes show only minor evidence of structural damage: some increase in intermyofibrillar space, with occasional areas of irregular SR disposition and an increase in frequency of smaller myofibrils, despite an increase of about 15 % in average myocyte cross sectional area. Z line streaming, a sign of myofibrillar stress, is limited and fairly rare. Immunolabeling with an anti-RyR2 antibody shows that RyR-positive foci located at the level of the Z lines are less frequent in mutant hearts. A dramatic decrease in the frequency and size of dyads, accompanied by a decrease in occupancy of the gap by RyR2, but without obvious alterations in location and general structure is a notable ultrastructural feature. The data suggest that the uneven distribution of dyads or calcium release sites within the cells resulting from an overall reduction in RyR2 content may contribute to the poor cardiac performance and early death of Ryr2 (ADA/ADA) mice. An unusual fragmentation of mitochondria, perhaps related to imbalances in free cytoplasmic calcium levels, accompanies these changes.

Cardiac hypertrophy associated with impaired regulation of cardiac ryanodine receptor by calmodulin and S100A1.

The cardiac ryanodine receptor (RyR2) is inhibited by calmodulin (CaM) and S100A1. Simultaneous substitution of three amino acid residues (W3587A, L3591D, F3603A; RyR2ADA) in the CaM binding domain of RyR2 results in loss of CaM inhibition at submicromolar (diastolic) and micromolar (systolic) Ca²âº, cardiac hypertrophy, and heart failure in Ryr2ADA/ADA mice. To address whether cardiac hypertrophy results from the elimination of CaM and S100A1 inhibition at diastolic or systolic Ca²âº, a mutant mouse was generated with a single RyR2 amino acid substitution (L3591D; RyR2D). Here we report that in single-channel measurements RyR2-L3591D isolated from Ryr2D/D hearts lost CaM inhibition at diastolic Ca²âº only, whereas S100A1 regulation was eliminated at both diastolic and systolic Ca²âº. In contrast to the ~2-wk life span of Ryr2ADA/ADA mice, Ryr2D/D mice lived longer than 1 yr. Six-month-old Ryr2D/D mice showed a 9% increase in heart weight-to-body weight ratio, modest changes in cardiac morphology, and a twofold increase in atrial natriuretic peptide mRNA levels compared with wild type. After 4-wk pressure overload with transverse aortic constriction, heart weight-to-body weight ratio and atrial natriuretic peptide mRNA levels increased and echocardiography showed changes in heart morphology of Ryr2D/D mice compared with sham-operated mice. Collectively, the findings indicate that the single RyR2-L3591D mutation, which distinguishes the effects of diastolic and systolic Ca²âº, alters heart size and cardiac function to a lesser extent in Ryr2D/D mice than the triple mutation in Ryr2ADA/ADA mice. They further suggest that CaM inhibition of RyR2 at systolic Ca²âº is important for maintaining normal cardiac function.

Deletion of HIF-1α partially rescues the abnormal hyaloid vascular system in Cited2 conditional knockout mouse eyes.

PURPOSE: Cited2 (CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2) is a member of a new family of transcriptional modulators. Cited2 null embryos exhibit hyaloid hypercellularity consisting of aberrant vasculature in the eye. The purpose of the study is to address whether abnormal lenticular development is a primary defect of Cited2 deletion and whether deletion of hypoxia inducible factor (HIF)-1α or an HIF-1α target gene, vascular endothelial growth factor (VEGF), could rescue abnormal hyaloid vascular system (HVS) in Cited2 deficient adult eyes. METHODS: Le-Cre specific Cited2 knockout (Cited2(CKO)) mice with or without deletion of HIF-1α or VEGF were generated by standard Cre-Lox methods. Eyes collected from six-eight weeks old mice were characterized by Real Time PCR and immunohistological staining. RESULTS: Cited2(CKO) mice had smaller lenses, abnormal lens stalk formation, and failed regression of the HVS in the adult eye. The eye phenotype had features similar to persistent hyperplastic primary vitreous (PHPV), a human congenital eye disorder leading to abnormal lenticular development. Deletion of HIF-1α or VEGF in Cited2 knockout eyes partially rescued the abnormal HVS but had no effect on the smaller lens and abnormal lens stalk differentiation. Intravitreal injection of Topotecan (TPT), a compound that inhibits HIF-1α expression, partially eliminated HVS defects in Cited2(CKO) lenses. CONCLUSIONS: Abnormal HVS is a primary defect in Cited2 knockout mice, resulting in part from dysregulated functions of HIF-1 and VEGF. The Cited2(CKO) mouse line could be used as a novel disease model for PHPV and as an in vivo model for testing potential HIF-1 inhibitors.

Protein kinase Cmu plays an essential role in hypertonicity-induced heat shock protein 70 expression.

Heat shock protein 70 (HSP70), which evidences important functions as a molecular chaperone and anti-apoptotic molecule, is substantially induced in cells exposed to a variety of stresses, including hypertonic stress, heavy metals, heat shock, and oxidative stress, and prevents cellular damage under these conditions. However, the molecular mechanism underlying the induction of HSP70 in response to hypertonicity has been characterized to a far lesser extent. In this study, we have investigated the cellular signaling pathway of HSP70 induction under hypertonic conditions. Initially, we applied a variety of kinase inhibitors to NIH3T3 cells that had been exposed to hypertonicity. The induction of HSP70 was suppressed specifically by treatment with protein kinase C (PKC) inhibitors (Gö6976 and GF109203X). As hypertonicity dramatically increased the phosphorylation of PKCmu, we then evaluated the role of PKCmu in hypertonicity-induced HSP70 expression and cell viability. The depletion of PKCmu with siRNA or the inhibition of PKCmu activity with inhibitors resulted in a reduction in HSP70 induction and cell viability. Tonicity-responsive enhancer binding protein (TonEBP), a transcription factor for hypertonicity-induced HSP70 expression, was translocated rapidly into the nucleus and was modified gradually in the nucleus under hypertonic conditions. When we administered treatment with PKC inhibitors, the mobility shift of TonEBP was affected in the nucleus. However, PKCmu evidenced no subcellular co-localization with TonEBP during hypertonic exposure. From our results, we have concluded that PKCmu performs a critical function in hypertonicity-induced HSP70 induction, and finally cellular protection, via the indirect regulation of TonEBP modification.

Local exposure of 849 MHz and 1763 MHz radiofrequency radiation to mouse heads does not induce cell death or cell proliferation in brain.

Even though there is no direct evidence to prove the cellular and molecular changes induced by radiofrequency (RF) radiation itself, we cannot completely exclude the possibility of any biological effect of mobile phone frequency radiation. We established a carousel-type exposure chamber for 849 MHz or 1763 MHz of mobile phone RF radiation to expose RF to the heads of C57BL mice. In this chamber, animals were irradiated intermittently at 7.8 W/kg for a maximum of 12 months. During this period, the body weights of 3 groups-sham, 849 MHz RF, and 1763 MHz RF-did not show any differences between groups. The brain tissues were obtained from 3 groups at 6 months and 12 months to examine the differences in histology and cell proliferation between control and RF exposure groups, but we could not find any change upon RF radiation. Likewise, we could not find changes in the expression and distribution of NeuN and GFAP in hippocampus and cerebellum, or in cell death by TUNEL assay in RF exposure groups. From these data, we conclude that the chronic exposure to 849 MHz and 1763 MHz RF radiation at a 7.8 W/kg specific absorption rate (SAR) could not induce cellular alterations such as proliferation, death, and reactive gliosis.

Molecular responses of Jurkat T-cells to 1763 MHz radiofrequency radiation.

PURPOSE: The biological effects of exposure to mobile phone emitted radiofrequency (RF) radiation are the subject of intense study, yet the hypothesis that RF exposure is a potential health hazard remains controversial. In this paper, we monitored cellular and molecular changes in Jurkat human T lymphoma cells after irradiating with 1763 MHz RF radiation to understand the effect on RF radiation in immune cells. MATERIALS AND METHODS: Jurkat T-cells were exposed to RF radiation to assess the effects on cell proliferation, cell cycle progression, DNA damage and gene expression. Jurkat cells were exposed to 1763 MHz RF radiation at 10 W/kg specific absorption rate (SAR) and compared to sham exposed cells. RESULTS: RF exposure did not produce significant changes in cell numbers, cell cycle distributions, or levels of DNA damage. In genome-wide analysis of gene expressions, there were no genes changed more than two-fold upon RF-radiation while ten genes change to 1.3 approximately 1.8-fold. Among ten genes, two cytokine receptor genes such as chemokine (C-X-C motif) receptor 3 (CXCR3) and interleukin 1 receptor, type II (IL1R2) were down-regulated upon RF radiation, but they were not directly related to cell proliferation or DNA damage responses. CONCLUSION: These results indicate that the alterations in cell proliferation, cell cycle progression, DNA integrity or global gene expression was not detected upon 1763 MHz RF radiation under 10 W/kg SAR for 24 h to Jurkat T cells.

Characterization of biological effect of 1763 MHz radiofrequency exposure on auditory hair cells.

PURPOSE: Radiofrequency (RF) exposure at the frequency of mobile phones has been reported not to induce cellular damage in in vitro and in vivo models. We chose HEI-OC1 immortalized mouse auditory hair cells to characterize the cellular response to 1763 MHz RF exposure, because auditory cells could be exposed to mobile phone frequencies. MATERIALS AND METHODS: Cells were exposed to 1763 MHz RF at a 20 W/kg specific absorption rate (SAR) in a code division multiple access (CDMA) exposure chamber for 24 and 48 h to check for changes in cell cycle, DNA damage, stress response, and gene expression. RESULTS: Neither of cell cycle changes nor DNA damage was detected in RF-exposed cells. The expression of heat shock proteins (HSP) and the phosphorylation of mitogen-activated protein kinases (MAPK) did not change, either. We tried to identify any alteration in gene expression using microarrays. Using the Applied Biosystems 1700 full genome expression mouse microarray, we found that only 29 genes (0.09% of total genes examined) were changed by more than 1.5-fold on RF exposure. CONCLUSION: From these results, we could not find any evidence of the induction of cellular responses, including cell cycle distribution, DNA damage, stress response and gene expression, after 1763 MHz RF exposure at an SAR of 20 W/kg in HEI-OC1 auditory hair cells.

Drebrin regulates angiotensin II-induced aortic remodelling.

Aims: The actin-binding protein Drebrin is up-regulated in response to arterial injury and reduces smooth muscle cell (SMC) migration and proliferation through its interaction with the actin cytoskeleton. We, therefore, tested the hypothesis that SMC Drebrin inhibits angiotensin II-induced remodelling of the proximal aorta. Methods and results: Angiotensin II was administered via osmotic minipumps at 1000 ng/kg/min continuously for 28 days in SM22-Cre+/Dbnflox/flox (SMC-Dbn-/-) and control mice. Blood pressure responses to angiotensin II were assessed by telemetry. After angiotensin II infusion, we assessed remodelling in the proximal ascending aorta by echocardiography and planimetry of histological cross sections. Although the degree of hypertension was equivalent in SMC-Dbn-/- and control mice, SMC-Dbn-/- mice nonetheless exhibited 60% more proximal aortic medial thickening and two-fold more outward aortic remodelling than control mice in response to angiotensin II. Proximal aortas demonstrated greater cellular proliferation and matrix deposition in SMC-Dbn-/- mice than in control mice, as evidenced by a higher prevalence of proliferating cell nuclear antigen-positive nuclei and higher levels of collagen I. Compared with control mouse aortas, SMC-Dbn-/- aortas demonstrated greater angiotensin II-induced NADPH oxidase activation and inflammation, evidenced by higher levels of Ser-536-phosphorylated NFκB p65 subunits and higher levels of vascular cell adhesion molecule-1, matrix metalloproteinase-9, and adventitial macrophages. Conclusions: We conclude that SMC Drebrin deficiency augments angiotensin II-induced inflammation and adverse aortic remodelling.

Crizotinib Inhibits Hyperpolarization-activated Cyclic Nucleotide-Gated Channel 4 Activity.

BACKGROUND: Sinus bradycardia is frequently observed in patients treated with crizotinib, a receptor tyrosine kinase inhibitor used for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). We investigated whether crizotinib could influence heart rate (HR) through direct cardiac effects. METHODS: The direct effect of crizotinib on HR was studied using ECG analysis of Langendorff-perfused mouse hearts. The whole-cell patch clamp technique was used to measure the effects of crizotinib on the hyperpolarization-activated funny current, If, in mouse sinoatrial node cells (SANCs) and hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) activity in HEK-293 cells stably expressing human HCN4. RESULTS: Crizotinib resulted in a dose-dependent reduction in HR in isolated intact mouse hearts with a half maximal inhibitory concentration (IC50) of 1.7 ± 0.4 µmol/L. Because ECG analysis revealed that crizotinib (0-5 µmol/L) resulted in significant reductions in HR in isolated mouse hearts without changes in PR, QRS, or QT intervals, we performed whole-cell patch clamp recordings of SANCs which showed that crizotinib inhibited If which regulates cardiac pacemaker activity. Crizotinib resulted in diminished current density of HCN4, the major molecular determinant of If, with an IC50 of 1.4 ± 0.3 µmol/L. Crizotinib also slowed HCN4 activation and shifted the activation curve to the left towards more hyperpolarized potentials. CONCLUSIONS: Our results suggest that crizotinib's effects on HCN4 channels play a significant role in mediating its observed effects on HR.

Effect of radiofrequency radiation exposure on mouse skin tumorigenesis initiated by 7,12-dimethybenz[alpha]anthracene.

PURPOSE: Although radiofrequency (RF) radiation is not considered mutagenic, it has been suggested as a promoter of tumorigenesis. To study if RF radiation has a tumor promoting effect, we exposed mice with skin tumorigenesis initiated by 7,12-dimethybenz[a]anthracene (DMBA) to RF radiation. MATERIALS AND METHODS: Eighty male ICR mice were subjected to a single DMBA application (100 microg/100 microl acetone/mouse) on shaved dorsal skin at the age of 7 weeks. After one week, the mice were randomized into four equal groups of 20 mice each: i.e., sham-, 849 MHz-, 1,763 MHz-exposed, and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated groups. The RF exposure was conducted at a whole body average specific absorption rate (SAR) of 0.4 W/Kg, for 2 cycles of 45 min exposure with a 15 min interval each day, 5 days a week for 19 weeks. The TPA-treated group served as a positive control for skin tumorigenesis and were administered TPA (4 microg/100 microl acetone/mouse) twice weekly without RF exposure. RESULTS: All mice were examined weekly at a macroscopic level. No skin tumors were observed in any groups except in the TPA-treated positive control group. TPA is known tumor promoter in DMBA-induced skin carcinogenesis and tumor incidence in the TPA treated group was 95%. At week 20 after DMBA initiation, skin tissues were analyzed immunohistochemically using anti-proliferating cell nuclear antigen (PCNA) antibody. No differences were observed by pathological examination or by PCNA staining between the sham- and the RF-exposed groups. The expression of cyclin D1 and c-fos were detected only in the tumorous skin tissues of the TPA-treated group. CONCLUSION: No evidence was found that RF radiation serves as a tumor promoter for skin tumors. Our data suggests that 849 MHz and 1,763 MHz RF radiations, similar to those emitted from mobile phones, do not have any promoting effect on skin tumor development in DMBA-initiated mice.

Subchronic exposure of hsp70.1-deficient mice to radiofrequency radiation.

PURPOSE: Heat shock protein 70 (HSP70) is one of the most inducible proteins to play a cytoprotective role under stressful conditions. Previously we generated hsp70.1-deficient mice to elucidate the in vivo function of HSP70 in detail. The renal tissues and embryonic fibroblasts of these mice were shown to be more vulnerable to hyperosmotic stress. Since RF (radiofrequency) energy has been suggested to be an environmental stressor, we carried out a study to determine whether sub-chronic RF exposure can cause constitutive induction of a stress response at a cellular and/or molecular level in hsp70.1-deficient mice due to repeated stimulation. MATERIALS AND METHODS: Eight-week-old hsp70.1-deficient mice were exposed twice daily for 45 min, with a 15 min interval, 5 days a week for 10 weeks. Whole-body average specific absorption rate was 0.4 W/Kg for fields of both 849 MHz and 1763 MHz. Major tissues were histopathologically analysed, and immunocytochemically evaluated for cell proliferative activity. Apoptosis was investigated by TdT-mediated dUTP nick-end labeling (TUNEL) assay. To determine whether RF radiation elicits a stress response, the expression level of heat shock proteins (HSP) and phosphorylation of the stress-activated kinases were also observed by western blots. RESULTS: No difference was observed in the histopathological analysis between sham- and RF-exposed mice. There was no evidence of increased proliferative and apoptotic activities. The levels of HSP90, HSP70, and HSP25 showed no obvious changes. RF exposure did not affect the phosphorylation status of the major stress-activated kinase (MAPK); extracellular signal-regulated kinase 1/2 (ERK1/2), C-Jun N-terminal kinase 1/2 (JNK1/2) or p38 MAPK. CONCLUSION: The hsp70.1-deficient mice did not show any significant changes in terms of cell proliferation, apoptosis, or stress response due to exposure of 849 or 1,763 MHz RF fields.

IL-6/STAT3 signaling in mice with dysfunctional type-2 ryanodine receptor.

Mice with genetically modified cardiac ryanodine receptor (Ryr2 (ADA/ADA) mice) are impaired in regulation by calmodulin, develop severe cardiac hypertrophy and die about 2 weeks after birth. We hypothesized that the interleukin 6 (IL-6)/signal transducer and activator of transcription-3 (STAT3) signaling pathway has a role in the development of the Ryr2 (ADA/ADA) cardiac hypertrophy phenotype, and determined cardiac function and protein levels of IL-6, phosphorylation levels of STAT3, and downstream targets c-Fos and c-Myc in wild-type and RyR2 (ADA/ADA) mice, mice with a disrupted IL-6 gene, and mice treated with STAT3 inhibitor NSC74859. IL-6 protein levels were increased at postnatal day 1 but not day 10, whereas pSTAT3-Tyr705/STAT3 ratio and c-Fos and c-Myc protein levels increased in hearts of 10-day but not 1-day old Ryr2 (ADA/ADA) mice compared with wild type. Both STAT3 and pSTAT3-Tyr705 accumulated in the nuclear fraction of 10-day old Ryr2 (ADA/ADA) mice compared with wild type. Ryr2 (ADA /ADA) /IL-6(-/-) mice lived 1.5 times longer, had decreased heart to body weight ratio, and reduced c-Fos and c-Myc protein levels. The STAT3 inhibitor NSC74859 prolonged life span by 1.3-fold, decreased heart to body weight ratio, increased cardiac performance, and decreased pSTAT-Tyr705/STAT3 ratio and IL-6, c-Fos and c-Myc protein levels of Ryr2 (ADA /ADA) mice. The results suggest that upregulation of IL-6 and STAT3 signaling contributes to cardiac hypertrophy and early death of mice with a dysfunctional ryanodine receptor. They further suggest that STAT3 inhibitors may be clinically useful agents in patients with altered Ca(2+) handling in the heart.

mTOR signaling in mice with dysfunctional cardiac ryanodine receptor ion channel.

Simultaneous substitution of three amino acid residues in the calmodulin binding domain (W3587A/L3591D/F3603A, ADA) of the cardiac ryanodine receptor ion channel (RyR2) impairs calmodulin inhibition of RyR2 and causes cardiac hypertrophy and early death of Ryr2ADA/ADA mice. To determine the physiological significance of growth promoting signaling molecules, the protein and phosphorylation levels of Ser/Thr kinase mTOR and upstream and downstream signaling molecules were determined in hearts of wild-type and Ryr2ADA/ADA mice. Phosphorylation of mTOR at Ser-2448, and mTOR downstream targets p70S6 kinase at Thr-389, S6 ribosomal protein at Ser-240/244, and 4E-BP1 at Ser-65 were increased. However, there was no increased phosphorylation of mTOR upstream kinases PDK1 at Ser-241, AKT at Thr-308, AMPK at Thr-172, and ERK1/2 at Thr-202/Tyr204. To confirm a role for mTOR signaling in the development of cardiac hypertrophy, rapamycin, an inhibitor of mTOR, was injected into wild-type and mutant mice. Rapamycin decreased mouse heart-to-body weight ratio, improved cardiac performance, and decreased phosphorylation of mTOR and downstream targets p70S6K and S6 in 10-day-old Ryr2ADA/ADA mice but did not extend longevity. Taken together, the results link a dysfunctional RyR2 to an altered activity of signaling molecules that regulate cardiac growth and function.

Inhibition of CaMKII does not attenuate cardiac hypertrophy in mice with dysfunctional ryanodine receptor.

In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca2+/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2ADA/ADA mice expressing the inhibitory peptide were not altered compared to control mice. In Ryr2ADA/ADA homozygous mice, AC3-I did not alter cardiac morphology, enhance cardiac function, improve sarcoplasmic reticulum Ca2+ handling, or suppress the expression of genes implicated in cardiac remodeling. The results suggest that CaMKII was not required for the rapid development of cardiac hypertrophy in Ryr2ADA/ADA mice.

Radiofrequency radiation does not induce stress response in human T-lymphocytes and rat primary astrocytes.

Heat shock proteins (HSPs) are rapidly induced by a variety of stressors, including heat shock, ethanol, heavy metals, UV, and gamma-radiation. Mitogen-activated protein kinases (MAPKs) are also involved in the stress transduction pathways in all eukaryotes. In this study, we attempted to determine whether radiofrequency (RF) radiation is able to induce a non-thermal stress response. Human T-lymphocyte Jurkat cells and rat primary astrocytes were exposed to 1763 MHz of RF radiation at an average specific absorption rate (SAR) of either 2 W/kg or 20 W/kg, for 30 min or 1 h. Temperature was completely controlled at 37 +/- 0.2 degrees C throughout the exposure period. The sham exposures were performed under exactly identical experimental conditions without exposure to RF radiation. We assessed alterations in the expression of HSPs and the activation of MAPKs in the RF-exposed cells. No detectable difference was observed in the expression levels of HSP90, HSP70, and HSP27. The phosphorylation status of MAPKs, extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal protein kinases (JNK1/2), or p38, did not change significantly. In order to determine whether RF radiation can promote the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on stress response, cells were exposed to RF radiation coupled with TPA treatment. When TPA alone was applied, the MAPKs were found to be phosphorylated in a dose-dependent manner. However, RF radiation did not result in any enhancement of TPA-induced MAPK phosphorylation. Neither TPA nor RF radiation exerted any detectable effect on the induction of HSPs. These results indicate that 1763 MHz RF radiation alone did not elicit any stress response, nor did it have any effect on TPA-induced MAPK phosphorylation, under our experimental conditions.