Roles of phospholipase D in phorbol myristate acetate‐stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10‐fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA‐stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA‐stimulated respiratory burst. Using 1‐butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA‐stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA‐stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA‐stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA‐stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2‐dioctanoyl‐sn‐glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA‐stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA‐stimulated respiratory burst.     

The mechanism and prevention of mitochondrial injury after exercise

Journal of Physiology and Biochemistry - With the development of society, physical activity has come to be an effective means by which people pursue good health to improve the quality of life....     

Mechanisms of regulation of phospholipase D1 by protein kinase Calpha

It has been suggested that protein-protein interaction is important for protein kinase C (PKC) alpha to activate phospholipase D1 (PLD1). To determine the one or more sites on PKCalpha that are involved in binding to PLD1, fragments containing the regulatory domain, catalytic domain, and C1-C3 domain of PKCalpha were constructed and shown to be functional, but they all failed to bind and activate PLD1 in vivo and in vitro. A C-terminal 23-amino acid (aa) deletion mutant of PKCalpha was also found to be inactive. To define the binding/activation site(s) in the C terminus of PKCalpha, 1- to 11-aa deletion mutants were made in this terminus. Deletion of up to 9 aa did not alter the ability of PKCalpha to bind and activate PLDl, whereas a 10-aa deletion was inactive. The residue at position 10 was Phe(663). Mutations of this residue (F663D and F663A) caused loss of binding, activation, and phosphorylation of PLD1, indicating that Phe(663) is essential for these activities. Time course experiments showed that the activation of PLD1 by PMA was much faster than its phosphorylation, and its activity decreased as phosphorylation increased with time. Staurosporine, a PKC inhibitor, completely inhibited PLD1 phosphorylation in response to 4beta-phorbol 12-myristate 13-acetate PMA and blocked the later decrease in PLD activity. The same results were found with the D481E mutant of PKCalpha, which is unable to phosphorylate PLD1. These results indicate that neither the regulatory nor catalytic domains of PKCalpha alone can bind to or activate PLD1 and that a residue in the C terminus of PKCalpha (Phe(663)) is required for these effects. The initial activation of PLD1 by PMA is highly correlated with the binding of PKCalpha. Although PKCalpha can phosphorylate PLD1, this is a relatively slow process and is associated with inactivation of the enzyme.     

Discovering Candidate Chromosomal Regions Linked to Kernel Size-Related Traits via QTL Mapping and Bulked Sample Analysis in Maize

Kernel size-related traits, including kernel length, kernel width, and kernel thickness, are critical components in determining yield and kernel quality in maize (Zea mays L.). Dissecting the phenotypic characteristics of these traits, and discovering the candidate chromosomal regions for these traits, are of potential importance for maize yield and quality improvement. In this study, a total of 139 F2:3 family lines derived from EHel and B73, a distinct line with extremely low ear height (EHel), was used for phenotyping and QTL mapping of three kernel size-related traits, including 10-kernel length (KL), 10-kernel width (KWid), and 10-kernel thickness (KT). The results showed that only one QTL for KWid, i.e., qKWid9 on Chr9, with a phenotypic variation explained (PVE) of 13.4% was detected between SNPs of AX-86298371 and AX-86298372, while no QTLs were detected for KL and KT across all 10 chromosomes. Four bulked groups of family lines, i.e., Groups I to IV, were constructed with F2:3 family lines according to the phenotypic comparisons of KWid between EHel and B73. Among these four groups, Group I possessed a significantly lower KWid than EHel (P = 0.0455), Group II was similar to EHel (P = 0.34), while both Group III and Group IV were statistically higher than EHel (P < 0.05). Besides, except Group IV exhibited a similar KWid to B73 (P = 0.11), KWid of Groups I to III were statistically lower than B73 (P < 0.00). By comparing the bulked genotypes of the four groups to EHel and B73, a stable chromosomal region on Chr9 between SNPs of AX-86298372 to AX-86263154, entirely covered by qKWid9, was identified to link KWid with the positive allele of increasing phenotypic effect to KWid from B73, similar to that of qKWid9. A large amount of enzyme activity and macromolecule binding-related genes were annotated within this chromosomal region, suggesting qKWid9 as a potential QTL for KWid in maize.     

1-Butanol interferes with phospholipase D1 and protein kinase Calpha association and inhibits phospholipase D1 basal activity

1-Butanol is commonly used as a substrate for phospholipase D (PLD) activity measurement. Surprisingly we found that, in the presence of 30 mM 1-butanol (standard PLD assay conditions), PLD1 activity in COS-7 cells was lost after incubation for 2 min. In contrast, in the presence of the protein kinase C (PKC) inhibitor staurosporine or dominant negative PKCalpha D481E, the activity was sustained for at least 30min. The binding between PLD1 and PKCalpha was also lost after 2 min incubation with 30 mM 1-butanol while staurosporine and D481E maintained the binding. 1-Butanol at 2 mM did not inhibit PLD1 basal activity or PLD1 binding to PKCalpha, and staurosporine and PKCalpha D481E produced a constant increase in PLD1 basal activity of 2-fold. These results indicate that 1-butanol is inhibitory to PLD1 activity by reducing its association with PKCalpha, and that the concentration of 1-butanol is an important consideration in assaying basal PLD1 activity.     

A point mutation at phenylalanine 663 abolishes protein kinase C alpha's ability to translocate to the perinuclear region and activate phospholipase D1

Previous research showed that protein kinase C alpha (PKC alpha) translocated to the perinuclear region and activated phospholipase D1, but the mechanism involved was not clear. Here, we provide evidence that Phe 663 (the 10th amino acid from C-terminus) of PKC alpha is essential for its translocation. A point mutation (F663D) completely blocked PKC alpha's binding to and activation of phospholipase D1. Further studies showed that deletion of the C-terminal nine amino acids of PKC alpha did not alter its translocation to the perinuclear region but deletion of the C-terminal 10 amino acids and the F663D mutation abolished this translocation. The F663D mutant was found to be resistant to dephosphorylation, which might account for its inability to translocate to the perinuclear region and activate PLD1, since dephosphorylation of PKC alpha is required for its relocation from plasma membrane to the perinuclear region.     

Histone Acetylation Regulation in Sleep Deprivation-Induced Spatial Memory Impairment

Sleep disorders negatively affect cognition and health. Recent evidence has indicated that chromatin remodeling via histone acetylation regulates cognitive function. This study aimed to investigate the possible roles of histone acetylation in sleep deprivation (SD)-induced cognitive impairment. Results of the Morris water maze test showed that 3 days of SD can cause spatial memory impairment in Wistar rats. SD can also decrease histone acetylation levels, increase histone deacetylase 2 (HDAC2) expression, and decrease histone acetyltransferase (CBP) expression. Furthermore, SD can reduce H3 and H4 acetylation levels in the promoters of the brain-derived neurotrophic factor (Bdnf) gene and thus significantly downregulate BDNF expression and impair the activity of key BDNF signaling pathways (pCaMKII, pErk2, and pCREB). However, treatment with the HDAC inhibitor trichostatin A attenuated all the negative effects induced by SD. Therefore, BDNF and its histone acetylation regulation may play important roles in SD-induced spatial memory impairment, whereas HDAC inhibition possibly confers protection against SD-induced impairment in spatial memory and hippocampal functions.     

H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib

Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.