A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.
The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO3. The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in Vmax.
The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.
Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.
Fusarium wilt and root-knot are important diseases of tomato. The use of chemical is becoming less appealing because of the health implications. Also, the chemicals required are often not within the reach of farmers in most of the developing part of the world. This research is aimed at finding an alternative mode of control. Tomato variety Roma VF inoculated with Meloidogyne and Fusarium were treated with 2 g/kg soil neem seed powder in the screenhouse and 2 Mg ha ? 1 in the field. An untreated and Furadan treated plot in the field served as control. Neem seed powder significantly reduced the disease severity of Fusarium and root-knot in both screenhouse and field. Results suggest the possible use of neem seed powder for control of the root-knot nematodes - Fusarium wilt disease complex. 相似文献
Cytotoxic T-lymphocyte (CTL) activity appears to play an important role in resolving hepatitis B virus (HBV) infection, and the ability to induce such responses remains an important goal for developing effective immunotherapeutics. A panel of recombinant retrovirus vectors expressing different forms of the HBV core antigen (HBcAg) or e antigen (eAg) were found to induce antigen-specific major histocompatibility complex-restricted CTL responses in both mice and macaques. In addition, a novel retrovirus vector expressing an HBcAg-neomycin phosphotransferase II (HBc-Neo) fusion protein [LHBc-NEO(6A3)], which allows the measurement of the anti-Neo antibody response as a means of directly tracking biological activity of the vector, was generated. Doses greater than 10(7) CFU were necessary to induce CTL responses in H-2(k) mice. Intramuscular injections with 10(8) CFU of the LHBc-NEO(6A3) retrovirus vector into rhesus monkeys induced HBc/eAg-specific antibody production and CD8+ CTLs. The CTL response from one of the two responder rhesus monkeys was directed against a 9-residue peptide, GELMTLATW, at positions 63 to 71 of the HBc/eAg sequence. The CTL response is long lived, being detectable as late as 16 weeks after immunization, and can be boosted upon reimmunization. The potent ability of recombinant retrovirus vectors to induce HBcAg- and eAg-specific CTL responses may prove beneficial as a therapeutic treatment for chronic hepatitis B infection. 相似文献
The purpose of this study was to evaluate the effect of formulation components on the in vitro skin permeation of microemulsion drug delivery system containing fluconazole (FLZ). Lauryl alcohol (LA) was screened as the
oil phase of microemulsions. The pseudo-ternary phase diagrams for microemulsion regions were constructed using LA as the
oil, Labrasol (Lab) as the surfactant and ethanol (EtOH) as the cosurfactant. The formulation which showed a highest permeation
rate of 47.15 ± 1.12 μg cm−2 h−1 and appropriate physicochemical properties was optimized as containing 2% FLZ, 10% LA, 20% Lab/EtOH (1:1), and 68% double-distilled
water (w/w). The efficiency of microemulsion formulation in the topical delivery of FLZ was dependent upon the contents of water and
LA as well as Lab/EtOH mixing ratio. It was concluded that the percutaneous absorption of FLZ from microemulsions was enhanced
with increasing the LA and water contents, and with decreasing the Lab/EtOH ratio in the formulation. Candida albicans was used as a model fungus to evaluate the antifungal activity of the best formula achieved, which showed the widest zone
of inhibition as compared to FLZ reference. The studied microemulsion formulation showed a good stability for a period of
3 months. These results indicate that the studied microemulsion formulation might be a promising vehicle for topical delivery
of FLZ. 相似文献
A wide variety of cell types, including immune cells, have been observed to frequently interact via transient, long-distance membrane connections [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. However, considerable heterogeneity in their structure, mode of formation and functional properties has emerged, suggesting the existence of distinct subclasses [18], [19], [20], [21]. Open-ended tunneling nanotubes allow for the trafficking of cytoplasmic material, e.g. endocytic vesicles, or the transmission of calcium signals [1], [8]. Closed-ended membrane nanotubes do not seamlessly connect the cytoplasm between two interacting cells and a junction exists within the nanotube or where the nanotube meets a cell body [4], [5], [7]. Recent live cell imaging suggested that membrane nanotubes between T cells could present a novel route for HIV-1 transmission [7], [22]. Here, we describe detailed protocols for observing membrane nanotubes and HIV-1 trafficking by live cell fluorescence microscopy. 相似文献