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801.
John O. Hui John Le Viswanatham Katta Robert Rosenfeld Michael F. Rohde Mitsuru Haniu 《Journal of Protein Chemistry》1996,15(4):351-358
Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors. 相似文献
802.
Mechanical effects of ET-1 in cardiomyocytes isolated from normal and heart-failed rabbits 总被引:1,自引:0,他引:1
Elizabeth J. Kelso Robert F. Geraghty Barbara J. McDermott Elisabeth R. Trimble D. Paul Nicholls Bernard Silke 《Molecular and cellular biochemistry》1996,157(1-2):149-155
Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca2+ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca2+ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n=7) or with saline (n=7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca2+ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 ± 11% (mean ± S.D.) in control cardiomyocytes and 33 ± 6% in heart-failed cells. However, there was no significant change in the EC50 obtained with ET-1 for healthy (0.31 ± 0.1 nM) and for failed cardiomyocytes (0.24 ± 0.1 nM). The effects of ET-1 on L-type Ca2+ channels were similar with a peak amplitude at 1 nM ET-1 of –3.26 ± 0.8 in control cardiomyocytes and –3.32 ± 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca2+ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.Recipient of Servier Investigator Award 相似文献
803.
Early after-depolarisations induced by noradrenaline may be initiated by calcium released from sarcoplasmic reticulum 总被引:1,自引:0,他引:1
We investigated the effect of 10–8 M noradrenaline (NA) on [Ca2+], and electrical activity of single myocytes of guinea-pig ventricular myocardium loaded with Indo 1-AM. Membrane potential was recorded by means of the patch electrode and patch amplifier set to the current clamp mode. Cells were stimulated at a rate of 30/min by 3 ms pulses of the current injected through the recording electrode. Superfusion of NA resulted in slight shortening of action potentials (APs), increase in rate of rise and amplitude of the respective Ca2+ transients, and appearance of secondary Ca2+ transients of two kinds: 1. appearing before repolarisation of AP and decay of the preceding Ca2+ transient were completed and 2. appearing between the APs. We named them early after-transients (EAT) and delayed after-transients (DAT), respectively. Without any additional intervention EATS caused some prolongation of APs duration and DATs resulted in subthreshold delayed after-depolarisations (DADS). When sarcolemmal K+ conductance was decreased by tetraethylammonium (TEA) in the patch electrode or 20 M BaCl2 in the Tyrode solution, EATs initiated early after depolarizations (EADs) and DATs initiated suprathreshold DADs triggering full-sized APs. Superfusion of 30.0 mM Na+ (replaced with LiCl) resulted in reduction of AP duration by -70% and appearance of DATs. Also, the frequent multiple oscillations of Ca 2+ concentration were often observed. Neither DATs nor the oscillations had any affect on electrical activity of the cells. Their electrogenicity could not be increased by TEA or 20.0 M Ba2+. EATs and DATs and their respective EADs and DADs could not be initiated by NA or low Na+ superfusion in the cells pretreated with 2 × 10–7 M thapsigargin, a selective blocker of Ca2+-ATPase of sarcoplasmic reticulum (SR). We conclude that in contrast to the current hypothesis, EADs can be initiated by Ca2+ released early in the cardiac cycle from the overloaded SR, and that electrogenicity of both types of Ca2+ oscillations critically depends on the sarcolemmal K+ conductance. 相似文献
804.
805.
Summary The glucoamylase gene from Lactobacillus amylovorus was cloned and expressed in Escherichia coli. A genomic DNA library from Lactobacillus amylovorus was prepared by partially digesting genomic DNA with EcoRI and ligating random fragments to the EcoRI digested cloning vector, pZErO-1.1. Three E. coli transformants expressing glucoamylase were identified using a probe prepared from the STA2 glucoamylase gene from Saccharomyces cerevisiae var. diastaticus. The physical maps of the recombinant plasmids were constructed. These plasmids contained inserts of about 5.2 Kb, 5.9 Kb and 6.4 Kb respectively. Temperature and pH optima of 45°C and 6.0, respectively, were obtained for both recombinant and purified wild type glucoamylases. Also, the enzymes were found to be thermolabile at temperatures above 50°C. 相似文献
806.
Yasmin H. Neggers Mary B. Dubard Robert L. Goldenberg Tsunenobu Tamura Kelley E. Johnston Rachel L. Copper John C. Hauth 《Biological trace element research》1996,55(1-2):127-135
Plasma zinc (Zn) concentrations were measured in 4376 indigent women (86% African-American), at a mean (±SD) gestational age
of 15 (±7.8) wk to determine the relationship between various maternal characteristics and plasma Zn levels during pregnancy.
Mean plasma Zn levels were lower in African-American women than in Caucasian women, in multiparous women than in primiparous
women, and in women with body weight >69.9 kg than in those with body weight ≤69.9 kg (p≤0.001 for each comparison). There were no significant differences related to maternal age, marital status, education, or
smoking habit. Multiple regression analysis, including maternal prepregnancy weight, race, age, parity, smoking habit, education,
and marital status indicated that race, parity, and pregnancy weight were significantly associated with maternal plasma Zn
levels, adjusted for gestational age. Maternal race was the best predictor of plasma Zn concentrations among the population
of pregnant women studied A significant proportion of variance in maternal plasma Zn levels remained unexplained after taking
into account various maternal characteristics. The reasons for lower plasma Zn levels in African-American women, compared
to Caucasian women, during pregnancy are unknown. 相似文献
807.
Six pesticides and two spray oils were tested against Polyphagotarsonemus latus. The chemicals were evaluated under laboratory conditions, requiring the development of a novel bioassay method, which is reported here. The pesticide toxicities fell into three distinct groups, namely abamectin, conventional pesticides and oils. The relative pesticide toxicities at the LC50 level were abamectin 4.9×10-8 g ai l-1, endosulfan 1.1×10-3 g ai l-1, fenpyroximate 2.3×10-3 g ai l-1, pyridaben 4.1×10-3 g ai l-1, tebufenpyrad 4.4×10-3 g ai l-1, dicofol 4.5×10-3 g ai l-1, petroleum spray oil 3.4×10-1 g ai l-1 and canola oil 4.1×10-1 g ai l-1. The calculation of the LC99.9 values allows for resistance monitoring in P. latus and the suggested discriminating concentrations are abamectin 1.0×10-4 g ai l-1; endosulfan, pyridaben and dicofol 1.0×10-1 g ai l-1 fenpyroximate and tebufenpyrad 5.0×10-1 g ai l-1. 相似文献
808.
Dissolved organic carbon (DOC) in Lake Fryxell, 10 streams flowing into the lake, and the moat surrounding the lake was studied to determine the influence of sources and biogeochemical processes on its distribution and chemical nature. Lake Fryxell is an amictic, permanently ice-covered lake in the McMurdo Dry Valleys which contains benthic and planktonic microbial populations, but receives essentially no input of organic material from the ahumic soils of the watershed. Biological activity in the water column does not appear to influence the DOC depth profile, which is similar to the profiles for conservative inorganic constituents. DOC values for the streams varied with biomass in the stream channel, and ranged from 0.2 to 9.7 mg C/L. Fulvic acids in the streams were a lower percentage of the total DOC than in the lake. These samples contain recent carbon and appear to be simpler mixtures of compounds than the lake samples, indicating that they have undergone less humification. The fulvic acids from just above the sediments of the lake have a high sulfur content and are highly aliphatic. The main transformations occurring as these fractions diffuse upward in the water column are 1) loss of sulfur groups through the oxycline and 2) decrease in aliphatic carbon and increase in the heterogeneity of aliphatic moieties. The fraction of modem14C content of the lake fulvic acids range from a minimum of 0.68 (approximately 3000 years old) at 15m depth to 0.997 (recent material) just under the ice. The major processes controlling the DOC in the lake appear to be: 1) The transport of organic matter by the inflow streams resulting in the addition of recent organic material to the moat and upper waters of the lake; 2) The diffusion of organic matter composed of relict organic material and organic carbon resulting from the degradation of algae and bacteria from the bottom waters or sediments of the lake into overlying glacial melt water, 3) The addition of recent organic matter to the bottom waters of the lake from the moat. 相似文献
809.
Woods Lake, in the Adirondack Mountains of New York, was the site of the Experimental Watershed Liming Study (EWLS) in which base addition was investigated as a method for mitigation of lake acidity. In an effort to predict the duration of effects, the treatment was simulated using the Integrated Lake-Watershed Acidification Study (ILWAS) model. To simulate terrestrial liming, calcite was applied to treated subcatchments as a rapidly weathering mineral in the upper horizon. Soil solution and lake outlet chemistry showed a response to calcite addition within four months of the start of the simulation. Calcium concentrations, acid neutralizing capacities (ANC), and pH increased in the upper soil layer and aluminum concentrations decreased in the upper three soil layers (0–70 cm). The response of ANC was delayed in lower soil layers due to proton production associated with aluminum hydrolysis. Moreover, soil water pH in the third soil layer decreased in response to calcite treatment due to the displacement of hydrogen ions by calcium added to the exchange complex. Calcium concentrations, ANC and pH increased and aluminum concentrations decreased in the simulated lake outlet. The modeled effects of calcite treatment on the soil and lake outlet chemistry were not as great as field observations. This was, in part, attributed to the model representation of the watershed, which did not include streams, ponds, or wetlands located in the treated subcatchments. Calcite applied to these saturated areas in the field readily dissolved, supplying ANC to lake water. Additionally, incorporation of calcite into a thick organic layer in the model diminished the possibility of dissolution by contact with overland flow. Observed concentrations of calcium, ANC, and pH in the outlet decreased after high values in the two years after treatment. Although the model failed to match observed short-term data, it may simulate the long-term response as calcium is transported through the soil. A long-term simulation of the model suggests that effects of base treatment will persist for at least 50 years. 相似文献
810.
The role of ethylene in anther culture of durum wheat ( Triticum durum Desf. cv. Ardente) was analyzed by testing the effects of 2-chloroethylphosphonic acid (ethrel) silver thiosulfate (Ag+ ), a -aminooxyacetic acid (AOA) and 1-aminocyclopropane-l-carboxylic acid (ACC) on microspore division observed after 21 days of culture and on development of calli estimated at day 45. The use of ethrel and Ag+ indicated a positive effect of ethylene on microspore division, whereas the use of AOA, and to a lesser extent ACC, snowed a negative effect. In contrast, the addition of ethrel or Ag+ indicated that ethylene inhibits the development of microspore-derived calli. AOA gave contradictory results. Ethylene production by anthers was about 7 pl anther−1 h−1 and decreased during culture. ACC content in the anthers was maximal at day 9, whereas malonyl ACC (MACC) increased sharply from day 0 to day 3 and then decreased. The addition of AOA or ACC to the culture medium decreased or increased, respectively, ethylene production of anthers and the ACC and/or MACC content, but at concentrations higher than those that modified the formation of calli. This formation seems to occur in two successive phases: induction and initiation of microspore division, which was promoted by ethylene, followed by callus development, which was inhibited by ethylene. 相似文献