The immunohistochemical localization of superoxide dismutase activity in the avian epithelial growth plate

Summary Superoxide dismutase (SOD) is a scavenger enzyme which catalyses the dismutation (reduction—oxidation) of the superoxide anion (O₂ ^–), a toxic free radical generated during normal cellular respiration. Light microscopy employing immunohistochemistry was utilized for localizing SOD activity in the chick epiphyseal cartilage. Antibodies to mammalian liver CuZn—SOD were prepared and the avidin—biotin—peroxidase technique (ABC complex) was utilized to localize activity for this enzyme in the growth plate cartilage. The localization of enzyme activity varied in accordance with the characteristic zonation pattern of the growth plate (zone of proliferation, zone of maturation, zone of cell hypertrophy and zone of matrix calcification). In the upper regions of the epiphyseal cartilage (the zones of proliferation and maturation), where the vascularity is poor and the oxygen tension low, SOD activity was localized within the chondrocytes. No extracellular activity was observed. However, in the lower regions of the growth plate (the zones of cell hypertrophy and matrix calcification), where both the vascularity and the oxygen tensions are increased, SOD activity was intense in both the chondrocytes and the surrounding extracellular matrix. Thus, the distribution of SOD enzyme activity in this tissue seems to vary in accordance with the level of oxygen present. The significance of the extracellular SOD activity, seen in the lower aspects of the growth plate cartilage, may indicate the sensitivity of matrix components, especially collagen, to toxic free radicals such as the superoxide anion.     

Biochemical characterization of a factor produced by trypomastigotes of Trypanosoma cruzi that accelerates the decay of complement C3 convertases

Infective- and vertebrate-stage trypomastigotes of Trypanosoma cruzi resist serum killing by the alternative complement pathway, whereas noninfective vector-stage epimastigotes, from which trypomastigotes derive, are serum-sensitive. This form of developmental preadaption is commonly observed in protozoan parasites, but its mechanisms are poorly understood. We have demonstrated previously that trypomastigotes spontaneously shed molecules which interfere with formation and accelerate the intrinsic decay of complement C3 convertases, a finding which may explain the evasion of complement lysis by trypomastigotes. We now describe the partial purification and characterization of the T. cruzi C3 convertase inhibitor from the supernatant of culture metacyclic and tissue culture trypomastigotes. Decay-accelerating activity for both classical and alternative pathway C3 convertases copurifies on anion-exchange fast protein liquid chromatography and chromatofocusing with 35S-labeled molecules of 87-93 kDa, pI 5.6-5.8. The labeled components are destroyed by papain and retained on concanavalin A-Sepharose, procedures which remove functional decay-accelerating activity from the supernatant. The 87-93-kDa components are immunoprecipitated by sera from patients chronically infected with T. cruzi, but not by antisera to any known regulatory proteins of the human complement cascade. Lytic activity for tissue culture trypomastigotes in chagasic sera is associated with antibody reactivity against the 87-93-kDa 35S-labeled components and with inhibition of decay-accelerating activity. The T. cruzi factor is the first developmentally regulated microbial complement inhibitor to be biochemically characterized.     

Kinetics of Choline Uptake into Isolated Rat Forebrain Microvessels: Evidence of Endocrine Modulation

The active uptake of [methyl-3H]choline into isolated rat brain microvessel suspension was studied as a likely guide to the transport of choline across the blood-brain barrier. The method consisted primarily of incubation of the suspension with a fixed concentration of labeled choline in the presence of increasing concentrations of unlabeled choline or any other inhibitor (I) of active uptake, defined as the difference in uptake at 37 degrees and 0 degrees C. From the linear regression of (1/V) against [I], the following values of Vmax (nmol g-1 min-1) and Km (microM) were obtained for choline: 2-month-old males, 10.6 +/- 3.8 and 6.1 +/- 0.9; 3-month old random females, 28.4 +/- 5.9 and 12.6 +/- 4.0; females at metaestrus, 17.8 +/- 10.3 and 8.3 +/- 5.0; at diestrus, 31.1 +/- 9.3 and 13.0 +/- 2.6; at proestrus, 54.9 +/- 2.2 and 14.0 +/- 1.5; at estrus, 19.2 +/- 2.2 and 2.6 +/- 1.7. The differences between males and random females (p less than 0.018) and between females at proestrus and estrus (p less than 0.005) are significant. It is suggested that these inter- and intrasex variations in choline uptake reflect a dynamic adjustment of supply in accordance with brain demand for choline at the time of assay. Hemicholinium-3 was an effective inhibitor of choline uptake, Ki = 14.0 +/- 8.5 microM; dimethylaminoethanol was much less effective; and imipramine had no measurable effect.     

Spatial distribution and turnover of root-derived carbon in alfalfa rhizosphere depending on top- and subsoil properties and mycorrhization

Aims

This study analyzed the extent to which root exudates diffuse from the root surface towards the soil depending on topsoil and subsoil properties and the effect of arbuscular mycorrhizal fungal hyphae on root-derived C distribution in the rhizosphere.

Methods

Alfalfa was grown in three-compartment pots. Nylon gauze prevented either roots alone or roots and arbuscular mycorrhizal fungal hyphae from penetrating into the rhizosphere compartments. ¹⁴CO₂ pulse labeling enabled the measurement of ¹⁴C-labeled exudates in dissolved (DOC) and total organic carbon (TOC) in the rhizosphere, distributed either by diffusion alone or by diffusion, root hair and hyphal transport.

Results

Root exudation and microbial decomposition of exudates was higher in the rhizosphere with topsoil compared to subsoil properties. Exudates extended over 28 mm (DOC) and 20 mm (TOC). Different soil properties and mycorrhization, likely caused by the low arbuscular mycorrhizal colonization of roots (13?±?4 % (topsoil properties) and 18?±?5 % (subsoil properties)), had no effect.

Conclusions

Higher microbial decomposition compensated for higher root exudation into the rhizosphere with topsoil properties, which resulted in equal exudate extent when compared to the rhizosphere with subsoil properties. Higher ¹⁴C activity used for labeling compared with previous studies enabled the detection of low exudate concentrations at longer distances from the root surface.