三七总皂甙和灯盏花素复方注射剂对结扎大鼠冠脉致心肌缺血的保护作用

采用结扎大鼠冠脉造成急性心肌缺血模型 ,观察了三七总皂甙和灯盏花素复方注射剂 (简称复方注射剂 )抗实验性心肌缺血作用。结果表明 :大鼠静脉注射复方注射剂 5 0mg/kg和 10 0mg/kg对急性心肌缺血均有一定程度的保护作用 ,在改善心电图S T段的上移、降低血清CPK、LDH方面两个剂量组均有显著作用 (P <0 .0 5 ,P <0 .0 1)以 10 0mg/kg组缩小心肌梗塞范围为显著 (P <0 .0 5 )。复方注射剂与丹参注射剂均能保护超氧化物歧化酶活性 (P <0 .0 5 ,P <0 .0 1) ,显著降低丙二醛含量 (P <0 .0 1)。复方注射剂两个剂量组均能显著增加前列环素合成 (P <0 .0 5 ,P <0 .0 1)。提示三七总皂甙和灯盏花素复方注射剂对心肌缺血的保护作用与抗脂质过氧化作用有关 ,其促进前列环素合成作用可能与其抗心肌缺血有关。     

Characterization of Protein Kinases in Apple and Grape During Fruit Development

The protein kinases were identified at different developmental stages of apple (Malus domestica L. Borth. cv. Red Starking) and grape (Vitis vinifera L. × V.lubrusca L. cv. Kyoho) fruit, and these protein kinases were found to be greatly activated by Ca2+. This demonstrated that calcium-dependent protein kinase plays an important role in apple and grape fruits. The results also showed that calmodulin (CaM) had no stimulation effect, but the CaM antagonists, such as calmidazolium, W7 and trifluoroacetic acid (TFA), had some inhibitory effects on the activities of this kind of protein kinase in both fruits, indicating that the detected protein kinases may be a kind of calmodulin-like domain protein kinase or calcium-dependent protein kinase (CDPK). There were distinct differences between apple and grape fruits, both in the characteristics and in the activity evolution patterns of the protein kinases during fruit development. The activity of calcium-dependent protein kinase was little changed in the three stages of apple fruit development, while in grape fruit the activity of this protein kinase was much higher in stage Ⅱ than those in stage Ⅰ and stage Ⅲ. The calcium-dependent protein kinase in apple fruits was strongly activated by Mn2+, and it was sensitive to heat treatment, but in grape fruits it was neither stimulated by Mn2+ nor sensitive to high temperature. Besides, phosphatidyl-serine (PS) had little effect on the activities of calcium-dependent protein kinase in grape fruits, but had stimulative effects in apple fruits. It was thus proposed that there might be a kind of calcium- and phosphatide-activated protein kinase, i.e., PKC, in apple fruits. The differences among the kinds, characteristics and the activity evolution patterns of protein kinases during apple and grape fruit development, suggest that the protein kinases may be involved in the process of fruit development.     

Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation

The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked de novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities in vitro. Whereas phosphorylation by casein kinase II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinase II (CaMKII) increased the binding activity 10–20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.     

"Muscle to meat" molecular events and technological transformations: the proteomics insight

Cellular death is characterized by a complex pattern of molecular events that depend on cell type. Specifically, muscle cells first undergo rigor mortis due to ATP depletion, and later, on the time scale of days, muscle fiber degradation due to proteolytic enzyme activity. In the present review, we will refer to proteomic investigations on the post-mortem evolution of the protein patterns of animal muscle cells. These studies, carried out with the application of either bottom-up or top-down methods, are relevant for understanding the biochemical reactions that i) convert muscle to meat, ii) are associated with meat aging and iii) impact on meat tenderness, a feature of significant commercial value. We also report on the proteomic investigations that have been made to analyze the transformation of meat in industrial processes. These studies are primarily aimed at identifying protein patterns and/or individual proteins diagnostic of the quality of the final product.     

Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase

The plant hormone abscisic acid (ABA) triggers production of reactive oxygen species (ROS) in guard cells via the AtrbohD and AtrbohF NADPH oxidases, leading to stomatal closure. The ABA-activated SnRK2 protein kinase open stomata 1 (OST1) (SRK2E/SnRK2.6) acts upstream of ROS in guard cell ABA signaling. Here, we report that OST1 phosphorylates Ser13 and Ser174 on AtrbohF. In addition, substitution of Ser174 to Ala results in a ∼40% reduction in the phosphorylation of AtrbohF by OST1. We also show that OST1 physically interacts with AtrbohF. These results provide biochemical evidence suggesting that OST1 regulates AtrbohF activity.

Structured summary

MINT-7260179, MINT-7260147, MINT-7260165: OST1 (uniprotkb:Q940H6) phosphorylates (MI:0217) ATRBOHF (uniprotkb:O48538) by protein kinase assay (MI:0424)MINT-7260208: OST1 (uniprotkb:Q940H6) and ATRBOHF (uniprotkb:O48538) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)     

Ionic channels and membrane hyperpolarization in human macrophages

Summary Microelectrode impalement of human macrophages evokes a transient hyperpolarizing response (HR) of the membrane potential. This HR was found to be dependent on the extracellular concentration of K⁺ but not on that of Na⁺ or Cl^–. It was not influenced by low temperature (12°C) or by 0.2mm ouabain, but was blocked by 0.2mm quinine or 0.2mm Mg²⁺-EGTA. These findings indicate that the HR in human macrophages is caused by the activation of a K⁺ (Ca²⁺) conductance. Two types of ionic channels were identified in intact cells by use of the patch-clamp technique in the cell-attached-patch configuration, low and high-conductance voltage-dependent K⁺ channels. The low-conductance channels had a mean conductance of 38 pS with Na⁺-saline and 32 pS with K⁺-saline in the pipette. The high-coductance channels had a conductance of 101 and 114 pS with Na⁺- and K⁺-saline in the pipette, respectively. Cell-attached patch measurements made during evocation of an HR by microelectrode penetration showed enhanced channel activity associated with the development of the HR. These channels were also high-conductance channels (171 pS with Na⁺- and 165 pS K⁺-saline in the pipette) and were voltage dependent. They were, however, active at less positive potentials than the high-conductance K⁺ channels seen prior to the microelectrode-evoked HR. It is concluded that the high-conductance voltage-dependent ionic channels active during the HR in human macrophages contribute to the development of the HR.     

Soluble 80-kd fragment of cell-CAM 120/80 disrupts cell-cell adhesion

Calcium-dependent cell adhesion molecules (CAMs) mediate intercellular adhesion in epithelial cells and in preimplantation mammalian embryos. One of these molecules, cell-CAM 120/80, is found on cells as a 120-kd membrane glycoprotein and as a soluble 80-kd species in conditioned culture medium [Damsky et al: Cell 34:455, 1983]. We have purified to homogeneity the soluble 80-kd fragment of cell-CAM 120/80 by using monoclonal antibody affinity chromatography. We have shown that the purified molecule can disrupt cell-cell adhesion in cultured epithelial cells, thus indicating that it is directly involved in the adhesive process. In addition, we have further characterized both the 120-kd cell-associated molecule and its 80-kd fragment, including N-terminal sequence analysis.