Cytokine production by dendritic cells genetically engineered to express IL-4: induction of Th2 responses and differential regulation of IL-12 and IL-23 synthesis

Dendritic cells (DCs) retrovirally transduced with IL-4 have recently been shown to inhibit murine collagen-induced arthritis and associated Th1 immune responses in vivo, but the mechanisms that underly these effects are not yet understood. In this report we demonstrate that IL-4-transduced DCs loaded with antigen led to lower T cell production of IFN-gamma, increased production of IL-4, and an attenuated, delayed type hypersensitivity response. We hypothesized that the ability of such DCs to regulate the Th1 immune response in vivo depends in part on their capacity to produce IL-12 and IL-23. Quantitative mRNA analysis revealed that IL-4-transduced DCs stimulated with CD40 ligand expressed higher levels of IL-12p35 mRNA, but lower levels of mRNA for IL-23p19 and the common subunit p40 found in both IL-12 and IL-23, compared with control DCs. These results, which indicate that expression of the IL-12 and IL-23 subunits is differentially regulated in IL-4-transduced DCs, were confirmed by ELISA of the IL-12 and IL-23 heterodimers. Thus, therapeutic suppression of Th1 -mediated autoimmunity (as recently shown in murine collagen-induced arthritis) and induction of Th2 responses in vivo by IL-4-transduced DCs occurs despite their potential to produce increased levels of IL-12, but could reflect, in part, decreased production of IL-23.     

Afterhyperpolarization–firing rate relation of turtle spinal neurons

This study addressed the afterhyperploarization–firing rate relationship of unanesthetized turtle spinal motoneurons and interneurons. The afterhyperploarization of their solitary action potential at rheobase was compared to that during the cells minimum and maximum firing rates. Like previous mammalian findings, afterhyperpolarization duration and area at rheobase were 32 and 19% less for high- versus low-threshold motoneurons. Contrariwise, maximum firing rate was two times less for the high-threshold group. Other new findings were that for high- versus low-threshold interneurons, afterhyperpolarization duration and area were 25 and 95% less, and maximum firing rate 21% higher for the high-threshold group. For combined motoneurons versus interneurons, there were no differences in afterhyperpolarization duration and area at rheobase, whereas maximum firing rate was 265% higher for the interneurons. For high-threshold motoneurons alone, there were significant associations between minimum firing rate and afterhyperpolarization duration and area measured at rheobase. In summary, this study showed that (1) the afterhyperploarization values of both turtle spinal motoneurons and interneurons at rheobase provided little indication of their corresponding values at the cells minimum and maximum firing states, and (2) the evolution of afterhyperploarization from rheobase to maximum firing state differed both qualitatively and quantitatively for motoneurons versus interneurons.     

Hepatobiliary excretion of dipyrrinone sulfonates in Mrp2-deficient (TR(-)) rats

The biliary excretion of the sodium salts of 8-(2-ethanesulfonic acid)-3-ethyl-2,7,9-trimethyl-1,10-dihydro-11H-dipyrrin-1-one (xanthosulfonic acid) and a fluorescent analogue (8-desethyl-N,N'-carbonyl-kryptopyrromethenone-8-sulfonic acid) was compared in Mrp2-deficient (TR(-)) and normal rats. Both organic anions were excreted rapidly in bile in Mrp2-deficient rats, but the biliary excretion of the fluorescent sulfonate was impaired relative to normal controls. The rat clearly has efficient Mrp2-independent mechanisms for biliary efflux of these anions that are not used by bilirubin or its mono- and diglucuronides.     

Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.     

Inhibited thrombins. Interactions with fibrinogen and fibrin.

Fibrin-monomer-Sepharose was used to study thrombin binding to fibrin and the role of the enzyme active centre in this interaction. Binding properties of preformed enzyme-inhibitor complexes, as well as inhibition of thrombin already adsorbed to fibrin monomer, were investigated. No apparent difference was found in binding properties of phenylmethanesulphonyl fluoride-, D-Phe-Pro-Arg-CH2Cl- and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide-inhibited thrombins. Also, the elution profile of phenylmethane-sulphonyl fluoride-inhibited thrombin from fibrinogen-Sepharose was identical with that of active thrombin from fibrin-monomer-Sepharose. Thus far the only low-Mr inhibitor that prevents thrombin from binding to fibrin monomer is pyridoxal 5'-phosphate. Preformed hirudin-thrombin complexes do not interact with fibrin. The extent to which the active centre of thrombin associated with fibrin is still accessible to substrates and inhibitors was also studied. Thrombin bound to fibrin hydrolyses a synthetic substrate at the same rate as the free enzyme. Water-soluble low-Mr inhibitors such as D-Phe-Pro-Arg-CH2Cl and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide can readily modify the active centre of the fibrin-associated enzyme, and the active centre is exposed to the degree that displacement of dansylarginine NN-(3-ethylpentane-1,5-diyl)amide by D-Phe-Pro-Arg-CH2Cl is possible without disturbing the binding. Hirudin disrupts the affinity between thrombin and fibrin. These data indicate that the active centre of thrombin associated with fibrin through extended binding is fully exposed and freely accessible. It is possible that extended binding may play a regulatory role in the activation of Factor XIII by thrombin, as well as inactivation of this enzyme by antithrombin III.     

Nitrogen fixation by groundnut and soyabean and residual nitrogen benefits to rice in farmers' fields in Northeast Thailand

Nitrogen fixation in groundnut and soyabean and the residual benefits of incoporated legume stover to subsequent rice crops were estimated in farmers' fields using¹⁵N-isotope methods. Three field experiments were conducted, two which examined N₂-fixation in groundnut by¹⁵N-isotope dilution using a non-nodulating groundnut as a reference crop and one in which N₂-fixation in two soyabean genotypes was compared using maize as the non-fixing reference crop. Groundnut fixed 72–77% of its N amounting to 150–200 kg N ha^-1 in 106–119 days and soyabean derived 66–68% of its N from N₂-fixation which amounted to 108–152 kg N ha^-1 under similar conditions. When legume stover was returned to the soil, there was a net contribution of N from N₂-fixing varieties of groundnut in all cases ranging from 13–100 kg N ha^-1, whilst due to the high % N harvest index in soyabean (87–88%) there was a net removal of N of 37–46 kg N ha^-1. In all cases if the legume stover was removed there was a net removal of N in the legume crop which ranged between 54 and 74 kg N ha^-1 in N₂-fixing varieties of groundnut and from 58 to 73 kg N ha^-1 in soyabean, whilst maize removed 66 kg N ha^-1 if its stover was returned and 101 kg N ha^-1 when the stover was removed. Growth of rice was improved in all cases where groundnut stover was returned resulting in increases in grain yield of 12–26% and increases in total dry matter production of 26–31%. Soyabean residues gave no increases in rice grain yield but increased total dry matter production by 12–20%. Rice accumulated more N in all cases where legume stover was returned to the soil, and N yields were larger in all cases after the N₂-fixing legumes than after the non-fixing reference crops. N difference estimates of the total residual N benefits from the N₂-fixing legumes ranged from 11–19 kg N ha^-1 after groundnut and 15–16 kg N ha^-1 after soyabean. The amounts of N estimated directly by application of¹⁵N-labelled stover amounted to 7.2–20.5 kg N ha^-1 with groundnut which represented recovery of 8–22% of the N added in the stover. In soyabean only 3.0–5.8 kg N ha^-1 was estimated to be recovered by¹⁵N-labelling which was 15–23% of the added N, whilst only 1.3 kg N ha^-1 (4% of the N added) was recovered by rice from the maize stover. An indirect¹⁵N-method based on addition of unlabelled stover to microplots where the soil had previously been labelled with¹⁵N gave extremely variable and often negative estimates of residual N benefits. Estimates of residual N from the added stover made by N difference calculations did not correspond with the estimates by direct¹⁵N-labelling in all cases and possible reasons for this are discussed.     

Grain legumes and green manures as pre-rice crops in Northeast Thailand

The loss of dry matter (ash corrected), nitrogen (N) and carbon (C) from residues of several tropical legume species was monitored using litter bags in the field over a three-month period in Northeast Thailand. This work was linked to an experiment in a farmers' field where the residual benefits of the same legume species grown before flooded rice were measured. Litter bags were incorporated in the flooded rice plots at the same time as residue incorporation in the field experiment. The species studied were Sesbania rostrata, Aeschynomene afraspera and a multi-purpose cowpea variety (Vigna unguiculata cv KVC-7). In the case of S. rostrata the breakdown of fresh and oven-dried residues and of residues buried at depths of 2–3 cm and 15 cm was also compared.Although the initial N and C concentrations were similar for all the residues they exhibited differing dry matter, N and C loss patterns. With Sesbania rostrata, 80% of the N was lost from the residues after 20 days, however, there was only a 40% decline in C and weight during the same period. The rate and amount of N loss from Aeschynomene afraspera residues was much less than with S. rostrata, declining by approximately 35% during the first 40 days. There were marked differences in rates of N loss from stem and leaves of A. afraspera indicating that monitoring the decomposition of stem and leaves combined can be misleading. In multi-purpose cowpea, loss patterns of dry matter, N and C were all similar and 50–65% was lost after 40 days burial. There was little difference between breakdown of fresh and oven-dried S. rostrata residues nor were there noticeable differences between residues incorporated superficially (2–3 cm) and buried at 15 cm. Although both %N and lignin:N ratios correlated well with weight and N loss from the residues, this was only the case when leaf and stem material were analyzed separately for A. afraspera.Despite the slower rate and smaller total amount of N released from the A. afraspera residues compared with the S. rostrata residues, a similar amount and proportion (around 20 kg N ha^-1 or 22–28%) of the N was recovered from both residues by a crop of rice planted at the time of residue incorporation. This suggests a considerably higher use efficiency by rice of the N released from the A. afraspera residues (approximately 40%) compared with that for S. rostrata (30%).