A semiautomated fluorometric determination of 5-hydroxyindoles in the nanogram range

Automated determinations of 5-hydroxytryptamine and its main metabolite, 5-hydroxyindoleacetic acid, have been described (Technicon autoanalyzer). The determinations are based on an extraction procedure from deproteinized tissue extracts or cerebrospinal fluid by means of butanolheptane mixtures. The indoles are transferred from the organic phase to a water phase and determined fluormetrically with the cysteine-o-phthaldialdehyde method. The method is highly sensitive: solutions containing 1 ng/ml can be reproducibly determined. Twenty samples per hour can be passed through the system. The determination of both 5-hydroxyindoles is performed with the same manifold.     

Purification of a delayed hypersensitivity-inducing protein from Listeria monocytogenes

Abstract Cell-envelope fragments were prepared from Listeria monocytogenes L242, serotype 4b. Delayed hypersensitivity (DH)-inducing proteins were extracted with deoxycholate and separated into two fractions by filtration through a Sephacryl S-200 column equilibrated with deoxycholate buffer. The second peak eluting from the Sephacryl column was fractionated using ion exchange chromatography on a DEAE Sepharose CL-6B column in the presence of 6 M urea. A purified 20 400-Da protein which induced DH against L. monocytogenes was obtained by isocratic elution. Three other DH-inducing fractions containing several protein bands were eluted by a gradient of potassium thiocyanate (KSCN) in urea buffer. Our results indicate that denaturing conditions can be employed for the fractionation and purification of DH inducing proteins from L. monocytogenes . In addition, it is suggested that the procedure described might also be useful for the purification of other antigens involved in cellular immune reactions.     

Effects of copper aspirinate and aspirin on tissue copper,zinc, and iron concentrations following chronic oral treatment in the adjuvant arthritic rat

Concentrations of copper, zinc, and iron were analyzed and compared in a number of tissues of adjuvant arthritic rats following 22 d of chronic treatment (per os) with either vehicle, aspirin or copper aspirinate, at doses of 100 mg/kg, 200 mg/kg, or 400 mg/kg. Such chronic treatment resulted in a negative balance in copper, zinc, and iron in many tissues. Among the tissues examined, liver and kidney exhibited the greatest changes in metal concentrations; brain and skeletal muscle exhibited the least. Arthritis-induced changes in the concentrations of all three metals in the liver were reversed upon treatment with aspirin. Treatment with copper aspirinate, on the other hand, resulted in an extremely high accumulation of copper in the liver. Arthritis-induced changes in copper, zinc, and iron concentrations in the pancreas and copper concentration in the plasma were generally not reversed upon treatment with either aspirin or copper aspirinate. Among the three metals examined, the degree of change observed as a result of drug treatments was greatest for iron and least for zinc. Finally, it appeared that the effects of aspirin and copper aspirinate on tissue metal concentrations were independent of the antiarthritic effects of these compounds.     

Effect of ammonium sulfate on the phytotoxicity, foliar uptake, and translocation of imazamethabenz in wild oat

Experiments were conducted in greenhouse, growth chamber, and laboratory conditions to determine the effect of ammonium sulfate [(NH₄)₂SO₄] on the phytotoxicity, foliar uptake, and translocation of imazamethabenz on wild oat. Rates of (NH₄)₂SO₄ up to 5% (w/v) applied with a greenhouse sprayer did not affect the phytotoxicity of the herbicide when the mix was applied at the one- to two-leaf stage. However, inclusion of 1 and 2% (NH₄)₂SO₄ increased the phytotoxicity of the herbicide when the mix was sprayed at the two- to three-leaf, or the three- to four-leaf stage. At 10%, (NH₄)₂SO₄ decreased the phytotoxicity of the sublethal dosage of the herbicide. When the herbicide was applied as individual drops to the growth chamber-grown plants, inclusion of (NH₄)₂SO₄ at 1% did not affect phytotoxicity as measured by shoot growth. The presence of (NH₄)₂SO₄ did not affect the amount of imazamethabenz retained by wild oat foliage, but it decreased [¹⁴C]imazamethabenz absorption, slightly antagonized acropetal translocation, and increased the basipetal translocation of [¹⁴C]imazamethabenz. It was concluded that application methods greatly modify the effect of (NH₄)₂SO₄ on imazamethabenz phytotoxicity. Herbicide absorption and translocation as determined by one method do not necessarily represent the absorption and translocation patterns when different application methods are used. Absorption and translocation were not the factors that were responsible for the observed effect of (NH₄)₂SO₄ on the herbicide phytotoxicity.Abbreviations SC suspension concentrate     

Efficient polymer-mediated delivery system for thiocyclam: Nanometerization remarkably improves the bioactivity toward green peach aphids

The unscientific application of synthetic pesticides has brought various negative effects on the environment, hindering the sustainable development of agriculture. Nanoparticles can be applied as carriers to improve pesticide delivery, showing great potential in the development of pesticide formulation in recent years. Herein, a star polymer (SPc) was constructed as an efficient pesticide nanocarrier/adjuvant that could spontaneously assemble with thiocyclam or monosultap into a complex, through hydrophobic association and hydrogen bonding, respectively, with the pesticide-loading contents of 42.54% and 19.3%. This complexation reduced the particle sizes of thiocyclam from 543.54 to 52.74 nm for pure thiocyclam, and 3 814.16 to 1 185.89 nm for commercial preparation (cp) of thiocyclam. Interestingly, the introduction of SPc decreased the contact angles of both pure and cp thiocyclam on plant leaves, and increased the plant uptake of cp thiocyclam to 2.4–1.9 times of that without SPc. Meanwhile, the SPc could promote the bioactivity of pure/cp thiocyclam against green peach aphids through leaf dipping method and root application. For leaf dipping method, the 50% lethal concentration decreased from 0.532 to 0.221 g/L after the complexation of pure thiocyclam with SPc, and that decreased from 0.390 to 0.251 g/L for cp thiocyclam. SPc seems a promising adjuvant for nanometerization of both pure and cp insecticides, which is beneficial for improving the delivery efficiency and utilization rate of pesticides.     

Crystal structure of a non-toxic mutant of heat-labile enterotoxin, which is a potent mucosal adjuvant.

Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 A resolution. Its structure appears to be essentially the same as the wild-type LT-I structure. The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I. The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.     

Protective Effects of Human Recombinant Mnsod in Adjuvant Arthritis and Bleomycin-Induced Lung Fibrosis

We have previously shown that human recombinant methionyl manganese superoxide dismutase (MnSOD) is more efficient than CuZnSOD as an anti-inflammatory agent in a model of acute inflammation (Carrageenan-induced pau edema). This effect was attributed to the prolonged half-life of MnSOD in blood (t_1/2 = 6 h vs. 10 min. respectively). In the present study, the two enzymes were compared in terms of their effectiveness in two systems: (I) Adjuvant-induced arthritis in rats, which is considered to be a model for the chronic situation of rheumatoid arthritis and (2) Bleomycin-induced lung fibrosis. which is a chronic situation believed to be mediated by oxygen free radicals.

Rats inflicted with adjuvant arthritis were treated during the period of maximal joint swelling (Days 15-21 after adjuvant injection) with MnSOD or CuZnSOD (12 to 50mg/kg, i.p. daily). MnSOD administration resulted in a 50-75% reduction of paw swelling, as well as inhibition of the elevation of serum globulins. A similar treatment with CuZnSOD gave merely marginal effects.

In the second system, lung fibrosis was induced in rats by intratracheal administration of bleomycin. MnSOD (50mg/kg, s.c.), administered 2 h before and then 2 and 4 days after bleomycin, markedly inhibited lung fibrosis, as evident from lung weight and collagen content measured by the 3rd week. By contrast, CuZnSOD administration did not give a significant effect. The results indicate that MnSOD is superior to CuZnSOD in the treatment of chronic inflammatory processes. In addition, they lend further support to the involvement of oxygen free radicals in bleomycin toxicity.     

Changes in plasma zinc,copper, iron,and hepatic metallothionein in adjuvant-induced arthritis treated with cyclosporin

The early changes in hepatic metallothionein (MT) and plasma zinc (Zn), copper (Cu), and iron (Fe) were investigated during the induction of adjuvant (AJ) arthritis in rats in conjunction with cyclosporin (CSA) treatment. Plasma Zn decreased after AJ injection (60% of control values at 8 h), and this was associated with a 4.5-fold increase in hepatic MT at 8 h. Plasma Zn was lowest at 16 h (40% of control), whereas hepatic MT concentrations increased to a maximum of 20-fold at 16 h. Changes in plasma Fe paralleled those of Zn, whereas plasma Cu levels were increased. Plasma metal and hepatic MT concentrations returned toward normal from d 1–7. At d 14, when marked paw swelling was apparent, hepatic MT and plasma Cu were again increased and plasma Zn decreased. Administration of CsA decreased MT induction in rats injected with AJ and also caused a marked recovery in plasma Zn and Fe levels. These changes were small but significant even in the early stages (up to 24 h) after AJ injection and were followed by a sustained improvement in all parameters, corresponding to the nonappearance of clinical arthropathy in CsA-treated rats. TNF-α and IL-6 production by peritoneal macrophages isolated from AJ-injected rats was significantly decreased by CsA treatment at d 7 and 14. The inhibition of hepatic MT induction during acute and chronic inflammation by cyclosporin emphasizes the role of the immune system in altered metal homeostasis in inflammation.