Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission

Oxygen-glucose deprivation (OGD) leads to depression of evoked synaptic transmission, for which the mechanisms remain unclear. We hypothesized that increased presynaptic [Ca²⁺]_i during transient OGD contributes to the depression of evoked field excitatory postsynaptic potentials (fEPSPs). Additionally, we hypothesized that increased buffering of intracellular calcium would shorten electrophysiological recovery after transient ischemia. Mouse hippocampal slices were exposed to 2 to 8 min of OGD. fEPSPs evoked by Schaffer collateral stimulation were recorded in the stratum radiatum, and whole cell current or voltage clamp recordings were performed in CA1 neurons. Transient ischemia led to increased presynaptic [Ca²⁺]_i, (shown by calcium imaging), increased spontaneous miniature EPSP/Cs, and depressed evoked fEPSPs, partially mediated by adenosine. Buffering of intracellular Ca²⁺ during OGD by membrane-permeant chelators (BAPTA-AM or EGTA-AM) partially prevented fEPSP depression and promoted faster electrophysiological recovery when the OGD challenge was stopped. The blocker of BK channels, charybdotoxin (ChTX), also prevented fEPSP depression, but did not accelerate post-ischemic recovery. These results suggest that OGD leads to elevated presynaptic [Ca²⁺]_i, which reduces evoked transmitter release; this effect can be reversed by increased intracellular Ca²⁺ buffering which also speeds recovery.     

A thermodynamic-kinetic analysis of the cytochrome P-450 heme pocket.

Spin state changes in the iron center of cytochrome P-450 during the catalytic cycle suggest alterations in the heme environment that insure proper substrate binding, an increase in redox potential, the formation of an active Fe-O complex, and the attack on the substrate. We used the spin state changes of the iron following physico-chemical perturbations, as an intrinsic probe of discrete changes around the heme, or of larger ones in the protein conformation. These environmental perturbations included temperature, solvent, substrate, and ionic environment. Aqueous and hydro-organic buffers provide complementary data and interpretations; the mixed solvent accommodates temperatures suitable for direct reaction rate measurements and amplified low to high spin transition. The results suggest that the group determining the heme spin state is influenced by the electrostatic potential created by several negative charges near the heme; the modulation of the spin state by various factors reflects the modulation of the electrostatic potential and of the internal paH value. Conformational changes of the whole protein are also indicated by the large entropy terms and their variation with experimental conditions.     

Identity of a cytosolic neutral cholesterol esterase in rat liver with the bile salt stimulated cholesterol esterase in pancreas

A neutral cholesterol esterase has been purified to homogeneity from the cytosolic fraction of rat liver. The 105,000 x g supernatant fraction of rat liver was applied to a DEAE-cellulose column to isolate a partially purified fraction of hepatic cholesterol esterase. Immunoblot analysis of the partially purified liver fraction with the anti-porcine pancreatic cholesterol esterase IgG demonstrated a single band with a molecular weight of 67,000. The hepatic protein was then isolated by immunoaffinity chromatography technique using a column constructed with antibodies prepared against the pancreatic cholesterol esterase. Characterization of the hepatic cholesterol esterase revealed that the hepatic enzyme shared antigenic epitopes with the pancreatic cholesterol esterase and was similarly activated by addition of bile salt such as taurocholate. Moreover, amino-terminal sequencing analysis of the hepatic cholesterol esterase showed an identical sequence with the pancreatic enzyme. Taken together, these results showed that the cholesterol esterases in the liver and the pancreas are very similar and possibly identical proteins.     

Kinetics of ultrastructural changes during electrically induced fusion of human erythrocytes

Summary The sequence of events during the electrically induced fusion of human erythrocytes was studied by rapid quench freeze-fracture electron microscopy. A single electric field pulse was used to induce fusion of human erythrocytes treated with pronase and closely positioned by dielectrophoresis. The electronic circuit was coupled to a rapid freezing mechanism so that ultrastructural changes of the membrane could be preserved at given time points. Pronase treatment enabled adjacent cells to approach each other within 15 nm during dielectrophoresis. The pulse caused a brief disruption of the aqueous boundaries which separated the cells. Within 100 msec following pulse application, the fracture faces exhibited discontinuous areas which were predominantly free of intramembranous particles. At 2 sec after the pulse, transient point defects attributed to intercellular contact appeared in the same membrane areas and replaced the discontinuous areas as the predominant membrane perturbation. At 10 sec after the pulse, the majority of the discontinuous areas and point defects disappeared as the intercellular distance returned to approximately 15 to 25 nm, except at sites of cytoplasmic bridge formation. Intramembranous particle clearing was observed at 60 sec following pulse application in discrete zones of membrane fusion.     

Phospho-regulated ACAP4-Ezrin Interaction Is Essential for Histamine-stimulated Parietal Cell Secretion

The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to the regulated HCl secretion. Our recent proteomic study revealed a protein complex of ezrin-ACAP4-ARF6 essential for volatile membrane remodeling (Fang, Z., Miao, Y., Ding, X., Deng, H., Liu, S., Wang, F., Zhou, R., Watson, C., Fu, C., Hu, Q., Lillard, J. W., Jr., Powell, M., Chen, Y., Forte, J. G., and Yao, X. (2006) Mol. Cell Proteomics 5, 1437–1449). However, knowledge of whether ACAP4 physically interacts with ezrin and how their interaction is integrated into membrane-cytoskeletal remodeling has remained elusive. Here we provide the first evidence that ezrin interacts with ACAP4 in a protein kinase A-mediated phosphorylation-dependent manner through the N-terminal 400 amino acids of ACAP4. ACAP4 locates in the cytoplasmic membrane in resting parietal cells but translocates to the apical plasma membrane upon histamine stimulation. ACAP4 was precipitated with ezrin from secreting but not resting parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction is abolished by phosphatase treatment and validated by an in vitro reconstitution assay using phospho-mimicking ezrin^S66D. Importantly, ezrin specifies the apical distribution of ACAP4 in secreting parietal cells because either suppression of ezrin or overexpression of non-phosphorylatable ezrin prevents the apical localization of ACAP4. In addition, overexpressing GTPase-activating protein-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and gastric acid secretion. Taken together, these results define a novel molecular mechanism linking ACAP4-ezrin interaction to polarized epithelial secretion.     

量天尺根部内生真菌的多样性

为了解不同量天尺（火龙果）品种根部内生真菌菌群组成及多样性,采集GHL-1、GHL-2、GHL-3、ML-1和DL 5个量天尺品种健康根部样品,进行内生真菌分离,采用形态观察和ITS序列分析相结合的方法进行鉴定、归类。共分离得到内生真菌菌株117株,总体分离率为25.71%,分别隶属于13个属,其中Trichoderma、Fusarium、Chaetomium和Phoma为量天尺内生真菌的优势种群,分别占总菌株数的24.79%、35.04%、10.26%和10.26%;不同量天尺品种内生真菌的结构和组成存在一定差异,GHL-2、GHL-3和DL 3个品种中分离频率最高的内生真菌类群为Fusarium,GHL-1和ML-1分离频率最高的类群为Trichoderma;多样性分析结果反映出不同量天尺品种内生真菌菌群的多样性指数、丰富度指数和均匀度指数水平存在差异,其中GHL-2的3项指数均为最高。表明品种差异对内生真菌的组成和多样性均有影响。     

Induction and characterization of tetraploids in poplar

Plant Cell, Tissue and Organ Culture (PCTOC) - Tetraploid poplar plants were induced by colchicine from tissue-cultured shoots; the induction efficiency varied with colchicine concentration....     

The Insufficiency Intake of Dietary Micronutrients Associated with Malnutrition-Inflammation Score in Hemodialysis Population

The relations between dietary micronutrient, nutritional status and inflammation in hemodialysis patients are still unclear. A cross-sectional study was performed in hemodialysis population. 75 hemodialysis patients from South China participated in the dietary and nutritional assessment. Clinical and dietary data were collected. Nutritional status was assessed by Malnutrition-Inflammation Score (MIS) in addition to related anthropometric measurements. And according to the MIS score, the whole hemodialysis patients were divided into normal nutrition group and malnutrition group. The results showed that mid arm circumference (MAC) negatively correlated with MIS (r = −0.425; P = 0.002). The area under the ROC curve (AUC) for MAC was 0.737 (0.614–0.859). Comparing with the normal nutritional group, lower dietary selenium (Se), copper (Cu), iodine (I) and manganese (Mn) intake were observed among patients with malnutrition (P<0.05). While no significant differences of diverse vitamins were found. In conclusion, MAC was effective indicator for assessing nutritional and inflammatory status (P<0.05). The reduction of dietary Se, Cu, I and Mn intake level may be alarming markers for malnutrition and inflammatory status in hemodialysis patients.