c-Cbl inhibition improves cardiac function and survival in response to myocardial ischemia.

BACKGROUND: The proto-oncogene Casitas b-lineage lymphoma (c-Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor and nonreceptor tyrosine kinases, resulting in their ubiquitination and downregulation. However, the function of c-Cbl in the control of cardiac function is currently unknown. In this study, we examined the role of c-Cbl in myocyte death and cardiac function after myocardial ischemia. METHODS AND RESULTS: We show increased c-Cbl expression in human ischemic and dilated cardiomyopathy hearts and in response to pathological stress stimuli in mice. c-Cbl-deficient mice demonstrated a more robust functional recovery after myocardial ischemia/reperfusion injury and significantly reduced myocyte apoptosis and improved cardiac function. Ubiquitination and downregulation of key survival c-Cbl targets, epidermal growth factor receptors and focal adhesion kinase, were significantly reduced in c-Cbl knockout mice. Inhibition of c-Cbl expression or its ubiquitin ligase activity in cardiac myocytes offered protection against H2O2 stress. Interestingly, c-Cbl deletion reduced the risk of death and increased cardiac functional recovery after chronic myocardial ischemia. This beneficial effect of c-Cbl deletion was associated with enhanced neoangiogenesis and increased expression of vascular endothelial growth factor-a and vascular endothelial growth factor receptor type 2 in the infarcted region. CONCLUSIONS: c-Cbl activation promotes myocyte apoptosis, inhibits angiogenesis, and causes adverse cardiac remodeling after myocardial infarction. These findings point to c-Cbl as a potential therapeutic target for the maintenance of cardiac function and remodeling after myocardial ischemia.

Soil warming-induced reduction in water content enhanced methane uptake at different soil depths in a subtropical forest.

Global warming can significantly impact soil CH4 uptake in subtropical forests due to changes in soil moisture, temperature sensitivity of methane-oxidizing bacteria (MOB), and shifts in microbial communities. However, the specific effects of climate warming and the underlying mechanisms on soil CH4 uptake at different soil depths remain poorly understood. To address this knowledge gap, we conducted a soil warming experiment (+4 °C) in a natural forest. From August 2020 to October 2021, we measured soil temperature, soil moisture, and CH4 uptake rates at four different soil depths: 0-10 cm, 10-20 cm, 20-40 cm, and 40-60 cm. Additionally, we assessed the soil MOB community structure and pmoA gene (with qPCR) at the 0-10 and 10-20 cm depths. Our findings revealed that warming significantly enhanced soil net CH4 uptake rate by 12.28 %, 29.51 %, and 61.05 % in the 0-10, 20-40, and 40-60 cm soil layers, respectively. The warming also led to reduced soil moisture levels, with more pronounced reductions observed at the 20-40 cm depth compared to the 0-20 cm depth. At the 0-10 cm depth, warming increased the relative abundance of upland soil cluster α (a type of MOB) and decreased the relative abundance of Methylocystis, but it did not significantly increase the pmoA gene copies. Our structural equation model analysis indicated that warming directly regulated soil CH4 uptake rate through the decrease in soil moisture, rather than through changes in the pmoA gene and MOB community structure at the 0-20 cm depth. In summary, our results demonstrate that warming enhances soil CH4 uptake at different depths, with soil moisture playing a crucial role in this process. Under warming conditions, the drier soil pores allow for better CH4 penetration, thereby promoting more efficient activity of MOB.

Functional type mediates the responses of root litter-driven priming effect and new carbon formation to warming.

Input of root litter can alter soil organic carbon (SOC) dynamics via causing priming effect (PE) on native SOC decomposition and forming new SOC. However, it is unknown how functional type mediates the root litter-driven PE and new C formation as well as their response to warming, which are of pivotal for soil C budget. We mixed litter segments of absorptive roots and transport roots from a Chinese fir (Cunninghamia lanceolata) plantation into isotopically distinct soil and incubated at 19°C (local mean annual temperature) and 23°C (warming by 4°C) for 210 days. Cumulative PE was calculated via integrating the instantaneous PE rates during the incubation. And the newly formed root litter-derived SOC (SOCrl) was calculated by measuring the δ13C value of soil at the end of incubation using a two-source mixed model. We found that absorptive roots with faster decomposition rates, caused significantly higher cumulative PE and SOCrl than transport roots. The microbial biomass and enzyme activities involved in C, N and P acquisition were significantly higher in the absorptive- than the transport roots addition treatment, indicating a higher level of microbial activation caused by absorptive roots. Although warming significantly increased the litter decomposition for both of functional types, while just significantly increased the PE of transport roots, indicating a root functional type dependent sensitivity of PE to warming. However, warming had no significant effect on SOCrl either for absorptive roots or for transport roots. As a consequence, warming relatively decreased the net SOC balance (difference between PE and SOCrl) in the transport roots addition treatment. Overall, our study highlights, for the first time, that functional type primarily mediates the response of root litter-driven PE to climate warming but not the new C formation, which may advance our understanding of SOC dynamics in Chinese fir plantation under climate change.

Regulatory domain determinants that control PKD1 activity.

The canonical pathway for protein kinase D1 (PKD1) activation by growth factor receptors involves diacylglycerol binding to the C1 domain and protein kinase C-dependent phosphorylation at the activation loop. PKD1 then autophosphorylates at Ser(916), a modification frequently used as a surrogate marker of PKD1 activity. PKD1 also is cleaved by caspase-3 at a site in the C1-PH interdomain during apoptosis; the functional consequences of this cleavage event remain uncertain. This study shows that PKD1-Δ1-321 (an N-terminal deletion mutant lacking the C1 domain and flanking sequence that models the catalytic fragment that accumulates during apoptosis) and PKD1-CD (the isolated catalytic domain) display high basal Ser(916) autocatalytic activity and robust activity toward CREBtide (a peptide substrate) but little to no activation loop autophosphorylation and no associated activity toward protein substrates, such as cAMP-response element binding protein and cardiac troponin I. In contrast, PKD1-ΔPH (a PH domain deletion mutant) is recovered as a constitutively active enzyme, with high basal autocatalytic activity and high basal activity toward peptide and protein substrates. These results indicate that individual regions in the regulatory domain act in a distinct manner to control PKD1 activity. Finally, cell-based studies show that PKD1-Δ1-321 does not substitute for WT-PKD1 as an in vivo activator of cAMP-response element binding protein and ERK phosphorylation. Proteolytic events that remove the C1 domain (but not the autoinhibitory PH domain) limit maximal PKD1 activity toward physiologically relevant protein substrates and lead to a defect in PKD1-dependent cellular responses.

c-Cbl ubiquitin ligase regulates focal adhesion protein turnover and myofibril degeneration induced by neutrophil protease cathepsin G.

The neutrophil-derived serine protease, cathepsin G (Cat.G), has been shown to induce myocyte detachment and apoptosis by anoikis through down-regulation of focal adhesion (FA) signaling. However, the mechanisms that control FA protein stability and turnover in myocytes are not well understood. Here, we have shown that the Casitas b-lineage lymphoma (c-Cbl), adaptor protein with an intrinsic E3 ubiquitin ligase activity, is involved in FA and myofibrillar protein stability and turnover in myocytes. Cat.G treatment induced c-Cbl activation and its interaction with FA proteins. Deletion of c-Cbl using c-Cbl knock-out derived myocytes or inhibition of c-Cbl ligase activity significantly reduced FA protein degradation, myofibrillar degeneration, and myocyte apoptosis induced by Cat.G. We also found that inhibition of the proteasome activity, but not the lysosome or the calpain activity, markedly attenuated FA and myofibrillar protein degradation induced by Cat.G. Interestingly, c-Cbl activation induced by Cat.G was mediated through epidermal growth factor receptor (EGFR) transactivation as inhibition of EGFR kinase activity markedly attenuated c-Cbl phosphorylation and FA protein degradation induced by Cat.G. These findings support a model in which neutrophil protease Cat.G promotes c-Cbl interaction with FA proteins, resulting in enhanced c-Cbl-mediated FA protein ubiquitination and degradation, myofibril degradation, and subsequent down-regulation of myocyte survival signaling.

p16INK4A flow cytometry of exfoliated cervical cells: Its role in quantitative pathology and clinical diagnosis of squamous intraepithelial lesions.

BACKGROUND: P16INK4A is a surrogate signature compensating for the specificity and/or sensitivity deficiencies of the human papillomavirus (HPV) DNA and Papanicolaou smear (Pap) co-test for detecting high-grade cervical squamous intraepithelial lesions or worse (HSIL+). However, traditional p16INK4A immunostaining is labour intensive and skill demanding, and subjective biases cannot be avoided. Herein, we created a high-throughput, quantitative diagnostic device, p16INK4A flow cytometry (FCM) and assessed its performances in cervical cancer screening and prevention. METHODS: P16INK4A FCM was built upon a novel antibody clone and a series of positive and negative (p16INK4A -knockout) standards. Since 2018, 24 100-women (HPV-positive/-negative, Pap-normal/-abnormal) have been enrolled nationwide for two-tier validation work. In cross-sectional studies, age- and viral genotype-dependent expression of p16INK4A was investigated, and optimal diagnostic parameter cut-offs (using colposcopy and biopsy as a gold standard) were obtained. In cohort studies, the 2-year prognostic values of p16INK4A were investigated with other risk factors by multivariate regression analyses in three cervicopathological conditions: HPV-positive Pap-normal, Pap-abnormal biopsy-negative and biopsy-confirmed LSIL. RESULTS: P16INK4A FCM detected a minimal ratio of 0.01% positive cells. The p16INK4A -positive ratio was 13.9 ± 1.8% among HPV-negative NILM women and peaked at the ages of 40-49 years; after HPV infection, the ratio increased to 15.1 ± 1.6%, varying with the carcinogenesis of the viral genotype. Further increments were found in women with neoplastic lesions (HPV-negative: 17.7 ± 5.0-21.4 ± 7.2%; HPV-positive: 18.0 ± 5.2-20.0 ± 9.9%). Extremely low expression of p16INK4A was observed in women with HSILs. As the HPV-combined double-cut-off-ratio criterion was adopted, a Youden's index of 0.78 was obtained, which was significantly higher than that (0.72) of the HPV and Pap co-test. The p16INK4A -abnormal situation was an independent HSIL+ risk factor for 2-year outcomes in all three cervicopathological conditions investigated (hazard ratios: 4.3-7.2). CONCLUSIONS: FCM-based p16INK4A quantification offers a better choice for conveniently and precisely monitoring the occurrence of HSIL+ and directing risk-stratification-based interventions.

Beta1-adrenergic receptors promote focal adhesion signaling downregulation and myocyte apoptosis in acute volume overload.

Numerous studies demonstrated increased expression of extracellular matrix (ECM) proteins and activation of focal adhesion (FA) signaling pathways in models of pressure overload-induced cardiac hypertrophy. However, little is known about FA signaling in response to volume overload where cardiac hypertrophy is associated with ECM loss. This study examines the role of beta1-adrenergic receptors (ß(1)-ARs) in FA signaling changes and myocyte apoptosis induced during acute hemodynamic stress of volume overload. Rats with eccentric cardiac hypertrophy induced after aorto-caval fistula (ACF) develop reduced interstitial collagen content and decreased tyrosine phosphorylation of key FA signaling molecules FAK, Pyk(2) and paxillin along with an increase in cardiac myocyte apoptosis. ACF also increased activation of PTEN, a dual lipid and protein phosphatase, and its interaction with FA proteins. ß(1)-AR blockade (extended-release of metoprolol succinate, 100mg QD) markedly attenuated PTEN activation, restored FA signaling and reduced myocyte apoptosis induced by ACF at 2days, but failed to reduce interstitial collagen loss and left ventricular dilatation. Treating cultured myocytes with ß(1)-AR agonists or adenoviral expression of ß(1)-ARs caused PTEN activation and interaction with FA proteins, thus leading to FA signaling downregulation and myocyte apoptosis. Adenoviral-mediated expression of a catalytically inactive PTEN mutant or wild-type FAK restored FA signaling downregulation and attenuated myocyte apoptosis induced by ß(1)-ARs. Collectively, these data show that ß(1)-AR stimulation in response to ACF induces FA signaling downregulation through an ECM-independent mechanism. This effect involves PTEN activation and may contribute to adverse cardiac remodeling and function in the course of volume overload.

Protein kinase D isoforms are activated in an agonist-specific manner in cardiomyocytes.

Protein kinase D (PKD) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PKD isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PKD1 and PKD2 in various cellular contexts. This study shows that PKD1 and PKD2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The α(1)-adrenergic receptor agonist norepinephrine selectively activates PKD1, thrombin and PDGF selectively activate PKD2, and endothelin-1 and PMA activate both PKD1 and PKD2. PKC activity is implicated in the α(1)-adrenergic receptor pathway that activates PKD1 and the thrombin- and PDGF-dependent pathways that activate PKD2. Endothelin-1 activates PKD via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PKD activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PKD substrates in cardiomyocytes. We show that overexpression of an activated PKD1-S744E/S748E transgene increases CREB-Ser(133) and cTnI-Ser(23)/Ser(24) phosphorylation, but agonist-dependent pathways that activate native PKD1 or PKD2 selectively increase CREB-Ser(133) phosphorylation; there is no associated increase in cTnI-Ser(23)/Ser(24) phosphorylation. Gene silencing studies provide unanticipated evidence that PKD1 down-regulation leads to a compensatory increase in PKD2 activity and that down-regulation of PKD1 (alone or in combination with PKD2) leads to an increase in CREB-Ser(133) phosphorylation. Collectively, these studies identify distinct roles for native PKD1 and PKD2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure.

[Effects of harvest residue management on soil organic nitrogen fractions in young Cunninghamia lanceolata plantation]. / 采伐剩余物不同处理方式对杉木幼林土壤有机氮组分的影响.

Different treatments of harvest residues will change the quantity and quality of soil organic matter, with direct or indirect effects on the composition and content of soil nutrient. Nitrogen is one of the most important soil nutrients. However, the response of soil organic nitrogen fractions to different harvest residue treatments is still unclear. In this study, harvest residue treatments, including harvest residue removed, residue retained and residue burnt, were set up after clear-cutting a 50-year-old mature Cunninghamia lanceolata forest in Sanming City, Fujian, China. The H2SO4 hydrolysis method was used to determine soil organic nitrogen fractions and their driving factors in the 0-10 cm and 10-20 cm soil layers after 5 years of harvest residue treatments. The results showed that residue retained treatment significantly enhanced the contents of soil organic nitrogen and its liable fractions. In the 0-10 cm soil layer, soil organic nitrogen content under residue retained treatment (3.36 g·kg-1) was 1.5 and 1.3 times as those of residue removed and residue burnt treatments, respectively. Residue retained treatment had the highest contents of labile nitrogen Ⅰ and Ⅱ fractions. In 10-20 cm soil layer, the contents of soil organic nitrogen and labile nitrogen Ⅱ fraction were also significantly higher in residue retained treatment (2.20, 0.73 g·kg-1) than that in residue removed and residue burnt treatments. The labile nitrogen index Ⅱ in residue retained treatment (33.9%) was significantly higher than in residue burnt treatment (26.1%). The contents of total carbon, dissolved organic carbon, dissolved organic nitrogen, microbial biomass under residue retained treatment were the highest in both soil layers. Compared with residue removed treatment, residue retained treatment significantly enhanced the abundance of soil bacteria (Gram-positive bacteria and Gram-negative bacteria) in 0-10 cm soil layer. In 10-20 cm soil layer, residue retained treatment had the highest content of fungi and the lowest content of actinomycetes. Pearson analysis showed that there were significant positive correlations of labile fractions of soil organic nitrogen with total carbon, dissolved organic carbon, dissolved organic nitrogen, microbial biomass carbon, microbial biomass nitrogen, bacteria (Gram-positive bacteria, Gram-negative bacteria), and fungi, and negative correlations with actinomycetes. It was concluded that the retention of harvest residue was beneficial to increase the content of soil organic nitrogen and labile fractions, improve soil biochemical properties and had a positive effect on soil microbial community composition. Retention of harvest residue was an effective management measure to maintain soil fertility and improve forest productivity.

Paclitaxel-Containing Extract Exerts Anti-Cancer Activity through Oral Administration in A549-Xenografted BALB/C Nude Mice: Synergistic Effect between Paclitaxel and Flavonoids or Lignoids.

Taxus yunnanensis is a paclitaxel-containing herb with traditional usage in cancer treatment, and its extract possesses great oral bioavailability of paclitaxel. However, it is elusive whether paclitaxel-containing extract (HDS-1) can exert anti-tumor effect through oral administration and how other components contribute to its efficacy. Therefore, we investigate the oral-route anti-tumor effect of HDS-1 in A549-bearing mice. HDS-1-derived flavonoids (HDS-2) and lignoids (HDS-3) are hypothesized to contribute to HDS-1's efficacy, and their effects of enhancing enterocytic absorption and cytotoxicity of paclitaxel are validated in 2 permeability experiments and apoptosis-related assay, respectively. In vivo, A549 growth is significantly inhibited by 86.1 ± 12.94% (P < 0.01) at 600 mg/kg of HDS-1 and 65.7 ± 38.71% (P < 0.01) at 200 mg/kg. HDS-2 and HDS-3 significantly reduce the efflux ratio of paclitaxel to 2.33 and 3.70, respectively, in Caco-2 permeability experiment and reduce paclitaxel reflux in MDCK-MDR1 experiment. Furthermore, HDS-2 and HDS-3 potentiated paclitaxel-induced cytotoxicity by 19.1-22.45% (P < 0.05) and 10.52-18.03% (P < 0.05), respectively, inhibited the expression of cyclinB1, Bcl-2, and pMCL-1, and increased the percentage of necrosis cell in the condition of paclitaxel exposure. Conclusively, paclitaxel-containing extracts exert anti-cancer effects through oral administration, and flavonoid and lignoids contribute to its anti-cancer effect through simultaneously improving enterocytic absorption of paclitaxel and the cytotoxic effect of paclitaxel.

Rapid detection of SARS-CoV-2 variants of concern by single nucleotide polymorphism genotyping using TaqMan assays.

We evaluated the performance of SARS-CoV-2 TaqMan real-time reverse-transcription PCR (RT-qPCR) assays (ThermoFisher) for detecting 2 nonsynonymous spike protein mutations, E484K and N501Y. Assay accuracy was evaluated by whole genome sequencing (WGS). Residual nasopharyngeal SARS-CoV-2 positive samples (N = 510) from a diverse patient population in New York City submitted for routine SARS-CoV-2 testing during January-April 2020 were used. We detected 91 (18%) N501Y and 101 (20%) E484K variants. Four samples (0.8%) were positive for both variants. The assay had nearly perfect concordance with WGS in the validation subset, detecting B.1.1.7 and B.1.526 variants among others. Sensitivity and specificity ranged from 0.95 to 1.00. Positive and negative predictive values were 0.98-1.00. TaqMan genotyping successfully predicted the presence of B.1.1.7, but had significantly lower sensitivity, 62% (95% CI, 0.53, 0.71), for predicting B.1.526 sub-lineages lacking E484K. This approach is rapid and accurate for detecting SARS-CoV-2 variants and can be rapidly implemented in routine clinical setting.

p66Shc links alpha1-adrenergic receptors to a reactive oxygen species-dependent AKT-FOXO3A phosphorylation pathway in cardiomyocytes.

p66Shc is an adapter protein that is induced by hypertrophic stimuli and has been implicated as a major regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. This study implicates p66Shc in an alpha(1)-adrenergtic receptor (alpha(1)-AR) pathway that requires the cooperative effects of protein kinase (PK)Cepsilon and PKCdelta and leads to AKT-FOXO3a phosphorylation in cardiomyocytes. alpha(1)-ARs promote p66Shc-YY(239/240) phosphorylation via a ROS-dependent mechanism that is localized to caveolae and requires epidermal growth factor receptor (EGFR) and PKCepsilon activity. alpha(1)-ARs also increase p66Shc-S(36) phosphorylation via an EGFR transactivation pathway involving PKCdelta. p66Shc links alpha(1)-ARs to an AKT signaling pathway that selectively phosphorylates/inactivates FOXO transcription factors and downregulates the ROS-scavenging protein manganese superoxide dismutase (MnSOD); the alpha(1)-AR-p66Shc-dependent pathway involving AKT does not regulate GSK3. Additional studies show that RNA interference-mediated downregulation of endogenous p66Shc leads to the derepression of FOXO3a-regulated genes such as MnSOD, p27Kip1, and BIM-1. p66Shc downregulation also increases proliferating cell nuclear antigen expression and induces cardiomyocyte hypertrophy, suggesting that p66Shc exerts an antihypertrophic action in neonatal cardiomyocytes. The novel alpha(1)-AR- and ROS-dependent pathway involving p66Shc identified in this study is likely to contribute to cardiomyocyte remodeling and the evolution of heart failure.

Protein kinase C-{delta} regulates the subcellular localization of Shc in H2O2-treated cardiomyocytes.

Protein kinase C-δ (PKCδ) exerts important cardiac actions as a lipid-regulated kinase. There is limited evidence that PKCδ also might exert an additional kinase-independent action as a regulator of the subcellular compartmentalization of binding partners such as Shc (Src homologous and collagen), a family of adapter proteins that play key roles in growth regulation and oxidative stress responses. This study shows that native PKCδ forms complexes with endogenous Shc proteins in H(2)O(2)-treated cardiomyocytes; H(2)O(2) treatment also leads to the accumulation of PKCδ and Shc in a detergent-insoluble cytoskeletal fraction and in mitochondria. H(2)O(2)-dependent recruitment of Shc isoforms to cytoskeletal and mitochondrial fractions is amplified by wild-type-PKCδ overexpression, consistent with the notion that PKCδ acts as a signal-regulated scaffold to anchor Shc in specific subcellular compartments. However, overexpression studies with kinase-dead (KD)-PKCδ-K376R (an ATP-binding mutant of PKCδ that lacks catalytic activity) are less informative, since KD-PKCδ-K376R aberrantly localizes as a constitutively tyrosine-phosphorylated enzyme to detergent-insoluble and mitochondrial fractions of resting cardiomyocytes; relatively little KD-PKCδ-K376R remains in the cytosolic fraction. The aberrant localization and tyrosine phosphorylation patterns for KD-PKCδ-K376R do not phenocopy the properties of native PKCδ, even in cells chronically treated with GF109203X to inhibit PKCδ activity. Hence, while KD-PKCδ-K376R overexpression increases Shc localization to the detergent-insoluble and mitochondrial fractions, the significance of these results is uncertain. Our studies suggest that experiments using KD-PKCδ-K376R overexpression as a strategy to competitively inhibit the kinase-dependent actions of native PKCδ or to expose the kinase-independent scaffolding functions of PKCδ should be interpreted with caution.

Enzyme stoichiometry indicates the variation of microbial nutrient requirements at different soil depths in subtropical forests.

Soil extracellular enzyme activities and associated enzymatic stoichiometry are considered sensitive indicators of nutrient availability and microbial substrate limitation. However, many of previous studies have been focusing on uppermost soil layer with a single enzyme as representative of the whole nutrient acquisition, leading to critical uncertainties in understanding soil nutrient availability and its relationship with microbial activities in deeper soils. In the current study, we investigated C-, N- and P-acquiring enzyme activities across a range of soil layers (0-10, 10-20, 20-40 and 40-60 cm), and examined the microbial C, N and P limitation in natural secondary forests (NSF) and Chinese fir (Cunninghamia lanceolata) plantation forests (CPF) in subtropical China. The results showed that microbial C and P co-limitation was detected in the two typical subtropical forests at all soil depths, rather than microbial N limitation. Microbial C and P limitation fluctuated along soil depth, but higher N was demanded by microbes in soil under 20 cm in both forests. The present results highlight the asymmetrical patterns of microbial nutrient limitation along the whole soil profile, and provide essential information in understanding nutrient limitations in deeper soils. These vertical and asymmetrical nutrient limitation patterns should be incorporated into future research studies priority.

Regulatory autophosphorylation sites on protein kinase C-delta at threonine-141 and threonine-295.

Protein kinase C-delta (PKCdelta) is a Ser/Thr kinase that regulates a wide range of cellular responses. This study identifies novel in vitro PKCdelta autophosphorylation sites at Thr(141) adjacent to the pseudosubstrate domain, Thr(218) in the C1A-C1B interdomain, Ser(295), Ser(302), and Ser(304) in the hinge region, and Ser(503) adjacent to Thr(505) in the activation loop. Cell-based studies show that Thr(141) and Thr(295) also are phosphorylated in vivo and that Thr(141) phosphorylation regulates the kinetics of PKCdelta downregulation in COS7 cells. In vitro studies implicate Thr(141) and Thr(295) autophosphorylation as modifications that regulate PKCdelta activity. A T141D substitution markedly increases basal lipid-independent PKCdelta activity; the PKCdelta-T141D mutant is only slightly further stimulated in vitro by PMA treatment, suggesting that Thr(141) phosphorylation relieves autoinhibitory constraints that limit PKCdelta activity. Mutagenesis studies also indicate that a phosphorylation at Thr(295) contributes to the control of PKCdelta substrate specificity. We previously demonstrated that PKCdelta phosphorylates the myofilament protein cardiac troponin I (cTnI) at Ser(23)/Ser(24) when it is allosterically activated by lipid cofactors and that the Thr(505)/Tyr(311)-phosphorylated form of PKCdelta (that is present in assays with Src) acquires as additional activity toward cTnI-Thr(144). Studies reported herein show that a T505A substitution reduces PKCdelta-Thr(295) autophosphorylation and that a T295A substitution leads to a defect in Src-dependent PKCdelta-Tyr(311) phosphorylation and PKCdelta-dependent cTnI-Thr(144) phosphorylation. These results implicate PKCdelta-Thr(295) autophosphorylation as a lipid-dependent modification that links PKCdelta-Thr(505) phosphorylation to Src-dependent regulation of PKCdelta catalytic function. Collectively, these studies identify novel regulatory autophosphorylations on PKCdelta that serve as markers and regulators of PKCdelta activity.

Reactive oxygen species decrease cAMP response element binding protein expression in cardiomyocytes via a protein kinase D1-dependent mechanism that does not require Ser133 phosphorylation.

Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser(133) in cardiomyocytes. Although CREB-Ser(133) phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser(133) phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser(133) phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

DUSP1 is controlled by p53 during the cellular response to oxidative stress.

p53 controls the cellular response to genotoxic stress through multiple mechanisms. We report here that p53 regulates DUSP1, a dual-specific threonine and tyrosine phosphatase with stringent substrate specificity for mitogen-activated protein kinase (MAPK). DUSP1 is a potent inhibitor of MAPK activity through dephosphorylation of MAPK. In a colon cancer cell line containing inducible ectopic p53, DUSP1 protein level is significantly increased upon activation of p53, leading to cell death in response to nutritional stress. In mouse embryo fibroblast cells, DUSP1 protein abundance is greatly increased after oxidative stress in a p53-dependent manner and also when apoptosis is triggered. We show that p53 induces the activity of a human DUSP1 regulatory region. Furthermore, p53 can physically interact with the DUSP1 regulatory region in vivo, and p53 binds to a 10-bp perfect palindromic site in this DUSP1 regulatory region. We show that overexpression of DUSP1 or inhibition of MAPK activity significantly increases cellular susceptibility to oxidative damage. These findings indicate that p53 is a transcriptional regulator of DUSP1 in stress responses. Our results reveal a mechanism whereby p53 selectively regulates target genes and suggest a way in which subgroups of those target genes might be controlled independently.

[Effects of harvest residue treatments on soil phosphorus fractions and availability in a young Chinese fir plantation]. / é‡‡ä¼å‰©ä½™ç‰©çš„å¤„ç†æ–¹å¼å¯¹æ‰æœ¨å¹¼æž—åœŸå£¤ç£·ç»„åˆ†åŠå ¶æœ‰æ•ˆæ€§çš„å½±å“.

Forest harvesting changes the quantity and quality of organic matter inputs into soil, and thus would alter soil nutrient content and availability. Phosphorus (P) is a key element affecting plant growth. The effects of harvest residue treatments on soil P fractions and availability had not yet been evaluated. In this study, harvest residue retainment (RR), residue removal (R) and residue burning (RB) treatments were manipulated after clear-cutting in a mature Chinese fir (Cunninghamia lanceolata) plantation at the Sanming Forest Ecosystem and Global Change Research Station in Fujian, China. This study focused on the dynamics of soil P fractions and their driving factors in the 0-10 cm and 10-20 cm soil layers after 4-year residue treatments. The results showed that, in RR treatment, the contents of easily-available P, moderately-available P and non-available P at the 0-10 cm soil layer were all significantly higher than those in R treatment, while the contents of moderately-available P and non-available P at the 10-20 cm soil layer was significantly higher than those in RB treatments. The ratios of soil organic carbon (C) to organic P (C:Po) in both layers were over 200 for all the three treatments, with ratios in RR treatment being significantly lower than those in RB and R treatments, indicating that RR could alleviate P limitation in this ecosystem. Moreover, results of the redundancy analysis showed that changes in P fractions were mainly affected by dissolved organic C, free Fe and noncrystalline amorphous Fe. The results suggested that soil organic P and available P were mainly from the decomposition of plant residues, which supported continuous P supply for plant growth. RR could enhance soil P content, thereby improve soil P availability and mitigate P limitation in Chinese fir plantation.

Alpha1-adrenergic receptors activate AKT via a Pyk2/PDK-1 pathway that is tonically inhibited by novel protein kinase C isoforms in cardiomyocytes.

AKT is a potent antiapoptotic kinase, but its role in the cardioprotective actions of alpha(1)-adrenergic receptors (ARs) remains uncertain, because alpha(1)-ARs typically induce little-to-no AKT activation in most cardiomyocyte models. This study identifies a prominent alpha(1)-AR-dependent AKT activation pathway that is under tonic inhibitory control by novel protein kinase Cs (nPKCs) in neonatal rat cardiomyocyte cultures. We also implicate Pyk2, Pyk2 complex formation with PDK-1 and paxillin, and increased PDK-1-Y373/376 phosphorylation as the mechanism that links alpha(1)-AR activation to increased AKT phosphorylation. nPKCs (which are prominent alpha(1)-AR effectors) interfere with this alpha(1)-AR-dependent AKT activation by blocking Pyk2/PDK-1/paxillin complex formation and PDK-1-Y373/376 phosphorylation. Additional studies used an adenoviral-mediated overexpression strategy to show that Pyk2 exerts dual controls on antiapoptotic PDK-1/AKT and proapoptotic c-Jun N-terminal kinase (JNK) pathways. Although the high nPKC activity of most cardiomyocyte models favors Pyk2 signaling to JNK (and cardiac apoptosis), the cardioprotective actions of Pyk2 through the PDK-1/AKT pathway are exposed when PKC or JNK activation is prevented. Collectively, these studies identify JNK and AKT as functionally distinct downstream components of the alpha(1)-AR/Pyk2 signaling pathway. We also implicate nPKCs as molecular switches that control the balance of signaling via proapoptotic JNK and antiapoptotic PDK-1/AKT pathways, exposing a novel mechanism for nPKC-dependent regulation of cardiac hypertrophy and failure.

Protease-activated receptor-1-mediated DNA synthesis in cardiac fibroblast is via epidermal growth factor receptor transactivation: distinct PAR-1 signaling pathways in cardiac fibroblasts and cardiomyocytes.

Proteases elaborated by inflammatory cells in the heart would be expected to drive cardiac fibroblasts to proliferate, but protease-activated receptor (PAR) function in cardiac fibroblasts has never been considered. This study demonstrates that PAR-1 is the only known PAR family member functionally expressed by cardiac fibroblasts and that PAR-1 activation by thrombin leads to increased DNA synthesis in cardiac fibroblasts. The increase in DNA synthesis induced by PAR-1 substantially exceeds the effects of other G protein-coupled receptor agonists in this cell type. PAR-1 stimulates phosphoinositide hydrolysis and mobilizes intracellular calcium via pertussis toxin (PTX)-sensitive and PTX-insensitive pathways. Activation of PAR-1 leads to an increase in Src, Fyn, and epidermal growth factor receptor (EGFR) phosphorylation, with EGFR receptor transactivation by Src family kinases the major mechanism for PAR-1-dependent activation of extracellular signal-regulated kinase, p38-mitogen-activated protein kinase, and protein kinase B. Activation of PAR-1 also leads to an increase in DNA synthesis. PAR-1 signaling is highly contextual in nature, inasmuch as PAR-1 activates extracellular signal-regulated kinase and only weakly stimulates protein kinase B via a pathway that does not involve EGFR transactivation in cardiomyocytes. PAR-1 responses in cardiac fibroblasts and cardiomyocytes are predicted to contribute importantly to remodeling during cardiac injury and/or inflammation.