EGTA与EDTA溶液去除根管壁玷污层能力的比较

目的：比较EGTA与EDTA去除根管玷污层的能力，为临床应用提供实验依据。方法：新鲜拔除24颗牙齿分为4组，即：15％EDTA、5％EGTA、10％EGTA、15％EGTA。实验组又分为：根管预备后冲洗(简称：甲组)，边扩边冲洗组(简称：乙组)。常规开髓、拔髓后15％EDTA、5％EGTA、10%EGTA、15％EGTA分别冲洗根管，剖开根管后在扫描电镜下观察根管壁玷污层及根管壁牙本质小管开口情况。结果：15％EDTA无论在根管上、中、下1／3均能较理想去除根管壁玷污层，牙本质小管开口清楚、密集。5％EGTA去除根管玷污层效果欠佳，而10％、15％EGTA效果较好。结论：15％EDTA，10%、15％EGTA均有较好去除根管壁玷污层的能力，而且冲洗效果与注射针头进入深度密切相关。     

Influence of adrenalin on sugar transport in soleus,a red skeletal muscle

The effect of adrenalin on the transport of the nonmetabolized sugar, 3-methylglucose, was studied in the isolated rat soleus. Basal sugar transport was inhibited and Na⁺ and K⁺ gradients were increased by 10^?8 to 10^?3 M adrenalin. Isoproterenol and phenylephrine had the same effects which were due to beta-adrenoceptor interaction. Adrenalin was also inhibitory under anoxic conditions and in the presence of palmitate and of low levels of insulin. External Na⁺, K⁺ or Ca²⁺ were not required for its effect and the data suggest that it involves a change in the apparent V_max of transport rather than in K_m. Inhibition of sugar influx was not always paralleled by lower internal Na⁺ levels, nor was it correlated with changes in ATP or G-6-P; this excludes several possible mechanisms for the effect. The failure of high adrenalin concentrations to stimulate sugar transport (as in diaphragm and heart muscle) is attributed to the high oxidative capacity of this red muscle.     

Myelin basic protein co-distributes with other PI(4,5)P2-sequestering proteins in Triton X-100 detergent-resistant membrane microdomains

The 18.5 kDa isoform of myelin basic protein (MBP) has recently been shown to sequester phosphatidylinositol-(4,5)-bis-phosphate (PI(4,5)P₂) in vesicular membranes in vitro, as do domains of other membrane- and cytoskeleton-associated proteins such as MARCKS (myristoylated alanine-rich C kinase substrate) and GAP-43 (growth-associated protein of 43 kDa), known collectively as “PI(4,5)P₂-modulins” [Musse et al., Biochemistry, 47 (2008) 10372–10382 (doi:10.1021/bi801302b)]. Here, we demonstrate co-localisation of MBP and MARCKS in primary rat oligodendrocytes, and co-distribution of MBP, MARCKS, and GAP-43 in lipid raft fractions recovered from Triton X-100 detergent-extracted isolated myelin and brain homogenates. The results lend further support to MBP's multifunctionality, particularly as an additional modulator of PI(4,5)P₂ availability in myelin.     

Hyperlipidemic mice present enhanced catabolism and higher mitochondrial ATP-sensitive K+ channel activity

BACKGROUND & AIMS: Changes in mitochondrial energy metabolism promoted by uncoupling proteins (UCPs) are often found in metabolic disorders. We have recently shown that hypertriglyceridemic (HTG) mice present higher mitochondrial resting respiration unrelated to UCPs. Here, we disclose the underlying mechanism and consequences, in tissue and whole body metabolism, of this mitochondrial response to hyperlipidemia. METHODS: Oxidative metabolism and its response to mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoK(ATP)) agonists and antagonists were measured in isolated mitochondria, livers, and mice. RESULTS: Mitochondria isolated from the livers of HTG mice presented enhanced respiratory rates compared with those from wild-type mice. Changes in oxygen consumption were sensitive to adenosine triphosphate (ATP), diazoxide, and 5-hydroxydecanoate, indicating they are attributable to mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) activity. Indeed, mitochondria from HTG mice presented enhanced swelling in the presence of K+ ions, sensitive to mitoK(ATP) agonists and antagonists. Furthermore, mitochondrial binding to fluorescent glibenclamide indicates that HTG mice expressed higher quantities of mitoK(ATP). The higher content and activity of liver mitoK(ATP) resulted in a faster metabolic state, as evidenced by increased liver oxygen consumption and higher body CO(2) release and temperature in these mice. In agreement with higher metabolic rates, food ingestion was significantly larger in HTG mice, without enhanced weight gain. CONCLUSIONS: These results show that primary hyperlipidemia leads to an elevation in liver mitoK(ATP) activity, which may represent a regulated adaptation to oxidize excess fatty acids in HTG mice. Furthermore, our data indicate that mitoK(ATP), in addition to UCPs, may be involved in the control of energy metabolism and body weight.     

Leukotrienes stimulate pepsinogen secretion from guinea pig gastric chief cells by a nitric oxide-dependent pathway

Leukotrienes (LTs) are involved in many inflammatory conditions including gastric damage induced by nonsteroidal anti-inflammatory drugs. Although LTs stimulate acid secretion, the effect they exert on pepsinogen secretion is unknown. The aim of this study was to investigate whether LTs stimulate pepsinogen secretion by isolated chief cells and to identify the intracellular messengers that mediate this action. Isolated chief cells were incubated with concentrations of LTB₄, LTC₄, LTD₄, or LTE₄ ranging from 0.1 pmol/L to 10 μmol/L, and pepsinogen release, intracellular calcium and inositol(1,4,5)-trisphosphate (IP₃) concentrations were measured. Nitric oxide generation was determined by the amount of citrulline generated during incubation. All four LTs caused a concentration-dependent stimulation of pepsinogen secretion with 50% effective concentration of 0.05-0.1 nmol/L and a dose-dependent increase in cytoplasmic free calcium and IP₃ concentration. The LTB₄ and LTD₄ antagonists caused selective, concentration-dependent inhibition of LTB₄- and LTD₄-induced pepsinogen secretion, calcium mobilization, and IP₃ generation. All four LTs increased NO generation, and the effect was inhibited by LTB₄ and LTD₄ antagonists and an NO synthase inhibitor N^G-monomethyl-l-arginine and reversed by l-arginine. N^G-monomethyl-l-arginine caused a 50%–60% reduction of LT-induced pepsinogen release. Each of the four LTs caused a fivefold increase in 5′-cyclic guanosine monophosphate. LTs are powerful stimulators of pepsinogen secretion in isolated chief cells and act via occupancy of specific cell-surface receptors.     

Biochemical and immunochemical studies on chick brain neurostenin

The KCl-extraction procedure employed for the isolation of actomyosin from muscle was applied to chick forebrain, the isolated fraction being termed chick neurostenin. This fraction was found to have Mg²⁺- or Ca²⁺ -ATPase activity, electrophoretic mobility on disc gel electrophoresis and immunochemical properties on immunodiffusion gels in general agreement with those reported for mammalian neurostenin. Immunofluorescence histochemistry indicated that antisera raised against chick neurostenin reacted specifically with central and peripheral nervous tissue, principally with neuronal and glial surfaces. Examination of preparative fractions by enzymic (Mg²⁺- or Ca²⁺ -ATPase) and immunochemical (microcomplement fixation) assays showed neurostenin to be associated with particulate materials. Staining profiles on sodium dodecyl sulphate-acrylamide gels confirmed chick neurostenin to be heterogenous and to contain lipid. Investigations of the chick neurostenin fraction for mechanochemical properties could not provide adequate support for an actomyosin-like nature.The results indicate that brain proteins with contractile or mechanochemical properties cannot be isolated with any significant degree of purification by applying a single cycle of extraction of chick brain into high ionic strength media (0.6M KG).     

Isolation of a polypeptide from the venom of Bungarus multicinctus that binds to ganglia and blocks the ganglionic transmission in mammals

A 15,000 dalton polypeptide purified from Bungarus multicinctus venom (which normally copurifies with alpha-bungarotoxin) was characterized biochemically and its biological effects were studied. This polypeptide, P15, had an aminoacid composition and molecular weight different from those of both alpha- and beta-bungarotoxin. It inhibited the ganglionic transmission in the guinea-pig hypogastric nerve-vas deferens preparation and did not block, even at very high concentrations, the neuromuscular transmission in the rat phrenic nerve-diaphragm preparation. In the same preparations alpha-bungarotoxin was unable to block the response at the ganglionic synapse while it was fully active in blocking the neuromuscular transmission. However, a pretreatment of the vas deferens preparation with alpha-bungarotoxin prevented the inhibitory effect of P15. 125I-Labeled P15 showed a specific and saturable binding to rat superior cervical ganglia homogenate and to a Torpedo postsynaptic membrane fraction. The binding of P15 to ganglia was inhibited by curare. The binding was Ca2+ dependent. The density of binding sites was of 300 fmol/mg of protein in the ganglion and 500 fmol/mg of protein in Torpedo membranes. The amount of P15-binding sites in ganglia was not modified by denervation, indicating that P15 binds to postsynaptic receptors. The binding of 125I-labeled P15, both in ganglia and Torpedo membranes, was inhibited by alpha-bungarotoxin. P15 had a Ca2+-dependent phospholipase A2 activity. Lowering Ca2+ concentration in incubation media affected the phospholipase A2 activity more than binding properties and inhibition of phospholipase activity with p-bromophenacyl bromide did not affect the activity of P15 on vas deferens preparation, suggesting that the phospholipase activity is not necessary for the activity of P15 on nicotinic receptors. Our results suggest that P15 toxin may be a specific and valuable probe for studying the ganglionic nicotinic receptor.     

Accumulation of [3H]fucose-labelled glycoproteins in the Golgi apparatus of dorsal root ganglion neurons during inhibition of fast axonal transport caused by exposure of the ganglion to Co2+-containing or Ca2+-free medium

Previous in vitro studies have established that Co2+-containing or Ca2+-free media interfere with the initiation of the fast axonal transport of proteins. The present study has used light- and electron-microscope radioautography to compare the distribution of [3H]fucose-labelled glycoproteins in neuronal cell bodies of control dorsal root ganglia and ganglia incubated for 16-17 h in Ca2+-free medium or in medium containing 0.18 mM Co2+. The radioautographic reaction in control cell bodies was diffusely scattered throughout the cytoplasm; grain counts revealed that 22% of the reaction was associated with elements of the Golgi apparatus and 78% was over other organelles and the remainder of the cytoplasm. In most experimental cell bodies, 78% of the silver grains were clustered over elements of the Golgi complex whereas other organelles and the remainder of the cytoplasm were comparatively much less labelled; structural alterations of the Golgi apparatus were also produced by the modified media. In parallel studies where the radioactivity in nerve trunks and ganglia was measured by liquid scintillation counting, it was found that the Ca2+-free medium and the Co2+-containing medium both reduced by approximately 80% the quantity of [3H]fucose-labelled glycoproteins which were carried by the fast axonal transport system; they did so without interfering with the incorporation of [3H]fucose into glycoproteins. The results indicate that in the presence of Co2+ or in the absence of Ca2+ the proteins which are destined for fast axonal transport accumulate at the Golgi apparatus of neuronal cell bodies. These results thus suggest that Ca2+ is required for proteins to leave the Golgi region in transit to the fast axonal transport system.     

The synaptically evoked late hyperpolarisation in hippocampal CA1 pyramidal cells is resistant to intracellular EGTA

It has been suggested that the late hyperpolarisation following synaptic activation of hippocampal CA1 pyramidal neurons is activated by calcium influx. This hypothesis was examined using microelectrodes containing EGTA. Intracellular injection of EGTA blocked the afterhyperpolarisation which normally followed cell firing produced by injection of a depolarising current or the ionophoresis of glutamate onto the apical dendrites. In contrast, the hyperpolarisation following synaptic activation was resistant to EGTA. The results suggest that this potential is not dependent on intracellular Ca2+. Other possible mechanisms are discussed.     

Immunoassay for wild-type protein in lymphocytes predicts germline mutations in patients at risk for hereditary colorectal cancer

Using colorectal cancer (CRC) as an example, we present the hypothesis that quantitative immunoassays for wild-type (full-length) proteins can be used to identify carriers of traits for hereditary diseases. In the case of hereditary CRC, this involves identifying individuals with germline mutations in a mismatch-repair (MMR) gene (mainly hMSH2 or hMLH1) or in the adenomatous polyposis coli (APC) gene. Because expression of wild-type protein should reflect wild-type gene dosage, we predicted that individuals harboring a germline mutation will have a reduction of approximately 50% in expression in lymphocytes of the corresponding full-length protein. In this pilot study, we tested lymphoblastoid cell lines that had been established from controls and individuals with, or at high risk for, hereditary CRC: 9 lines from healthy, unaffected individuals; 4 from affected members in familial adenomatous polyposis families (with known germ-line APC mutation); 42 from CRC patients in our Familial CRC Registry (increased risk of hereditary nonpolyposis colon cancer as assessed by family history, age at adenoma or carcinoma diagnosis, and other clinical criteria). For MSH2 and MLH1 we used western blots; for APC we used immunoprecipitation. All familial adenomatous polyposis lines had about 50% less immunoprecipitable full-length APC protein. Some cell lines (7 of 42) from Familial CRC Registry patients showed on western blots a reduction (mean 46%) in either MSH2 or MLH1 (relative to the other protein). All 7 subsequently were proved to contain a germline MMR mutation. We conclude that (1) because most of the expected CRC-causing germ line mutations are truncation-causing, immunoassays for wild-type protein should be able to identify most individuals with hereditary CRC-causing traits; (2) these assays, which are more practical and inexpensive than current mutation-detecting tests for hereditary CRC traits, have the potential for commercial development into broad-based population screens of high-risk patients and their families and the potential to save both lives and health-care dollars; (3) this strategy may be useful for other hereditary cancers and even other hereditary diseases; (4) our approach has the potential to greatly benefit public-health programs for cancer control.