A quantitative analysis of rat adrenal chromaffin tissue: Morphometric analysis at tissue and cellular level correlated with catecholamine content

A morphometric analysis of normal Wistar rat adrenal medulla following perfusion fixation and Araldite embedding, was correlated with catecholamine levels on fresh tissue, measured by high-performance liquid chromatography. The mean volume of whole adrenal is 13.2 mm³ and the mean medullary volume 1.3mm³. Volume density estimates showed that the medulla is composed of 63% chromaffin tissue with an adrenaline to noradrenaline storing cell ratio of 4.4:1. The vasculature occupies 20%, neuronal tissue 5% and interstitial tissues 12% of the medulla. A comparison was made of cell volumes, cell numbers and volume and surface density estimates of cytoplasmic organdies in adrenaline and noradrenaline storing cells. The mean cell volume of adrenaline storing cells at 1300 μm³ is larger than that of noradrenaline storing cells at 980 μm³. A single adrenal medulla contains4.4−5.7 × 10⁵ adrenaline cells and1.5−1.9 × 10⁵ noradrenaline cells. Chromaffin granules account for approximately 30% of the volume of the cytoplasm; the numerical density of granules at different sites in the cell was calculated for adrenaline cells. The volume density of mitochondria (4%) and the surface density of mitochondrial membranes (the ratio of outer to inner membrane being approximately 1:2.3) were similar in both cell types. Rough endoplasmic reticulum was the only organelle to show a significant difference in volume and surface density between the two cell types. Adrenaline storing cells have stacks of rough endoplasmic reticulum which have two to three times the surface and volume densities of that found diffusely scattered throughout noradrenaline cells. The adrenaline content of an adrenaline storing cell is0.14 × 10⁻⁶ μM and that of a granule 3.0 × 10⁻¹² or3.8 × 10⁻¹² μ moles depending on the method of calculation. The noradrenaline content of noradrenaline storing cells can only be calculated on the assumption that all noradrenaline is stored in this cell type though it is likely that some is contained within adrenaline cells. Based on this assumption the noradrenaline content is0.17 × 10⁻⁶μ moles per cell and5 × 10⁻¹² μ moles per granule. The present study provides baseline morphometric data on the rat adrenal medulla at tissue and cellular level correlated with amine levels in adrenaline and noradrenaline storing cells and granules.     

A method for analysing the clonal precursors of concanavalin A-induced suppressor cells

Spleen cells from 3 different strains of mice (C57 (H-2^b), CBA (H-2^k) and BALB/c (H-2^d)) were stimulated in vitro with different concentrations of concanavalin A (CA) for 48 h. This resulted in the production of cells capable of inhibiting the generation of alloantigen-specific cytotoxic T lymphocytes (CTL) in a mixed lymphocyte culture (MLC). 1 μg/ml was an effective concentration of CA to induce C57 and BALB/c suppressor cells (SC), but 5 μg/ml CA was required to induce CBA SC. SC precursors (SC-P) were shown to be radiosensitive and the results suggest that SC themselves may be radiosensitive. SC were effective in the presence of added interleukin-2 (IL-2).

SC were then induced at limit dilution in microwells in a volume of 25 μl. A MLC (200 μl) was then (after 48 h) added to each microwell. This resulted in a dilution of the concentration of CA to a level below which it was effective at inducing suppression. Cytotoxicity was then assessed 7 days later. It was thus possible to analyse SC-P at the clonal level and estimate their frequency. The frequency of C57 splenic SC-P (active against a C57 anti-BALB/c MLC) was 14.4 × 10⁻⁶, the frequency of CBA splenic SC-P (active against a CBA anti-BALB/c MLC) was 92.3 × 10⁻⁶, and the frequency of BALB/c splenic SC-P (active against a BALB/c anti-CBA MLC) was 15.8 × 10⁻⁶. It was possible to analyse SC-P at a clonal level whether or not the MLC contained added IL-2. SC and SC-P were shown to be sensitive to anti-Thy-1 and complement.     

Human extracellular proteins display a different pattern of local sequence similarity with the four classes of human T-cell receptor V-regions than foreign proteins and human intracellular proteins: a preliminary report

A pool of 110 randomly selected/generated amino acids sequences was used to perform specific local sequence similarity alignment analysis with the pool of 279 reported sequences of human T-cell receptor (TCR) V-regions. The 110 analyzed sequences were divided, according to their origin and nature, into six protein groups, as: human intracellular (hi), extracellular/transmembrane (he) and extracellular adhesive matrix (ha) proteins, ‘average' human proteins (hum), proteins of non-human origin (nhum) and randomly generated quasi-protein sequences (r). These sequences were decomposed into all their overlapping 11-mer segments, generating a total of 56 836 derived peptides (at least 8000 per group). Each derived peptide was aligned with the 279 human TCR V-regions and assigned to the category (-like, β-like, γ-like or δ-like) corresponding to the class (V, Vβ, Vγ or Vδ) of the V-region encompassing the most similar segment, as determined by the performed similarity-search. The six protein groups were found to differ significantly in their distribution of derived peptides among the four categories. According to the binomial tests results, human proteins from the extracellular compartment (he, ha) comprise a higher proportion of δ-like segments (P=2.3×10⁻² and P<10⁻⁸, respectively) than the ‘average' human proteins (hum). In addition, and in accordance with this finding, proteins that are normally not found in that topological compartment comprise a lower proportion of δ-like peptides (P=1.4×10⁻⁵ and P<10⁻⁸ for groups nhum and hi, respectively) than the ‘average' human proteins (hum). In contrast, these proteins comprise a higher proportion of γ-like segments (P=8.3×10⁻³, P=1.4×10⁻³ and P=1.7×10⁻⁴, for groups r, nhum and hi, respectively) than the ‘average' human proteins (hum). These findings indicate significant differences between proteins encountered in the extracellular compartment—that are normally immunologically tolerated—and those the presence of which is usually non-tolerated. The results suggest that the discrimination and the reaction of the human immune network to proteins found in the extracellular compartment correlate with the proteins' pattern of preferential local sequence similarity with the Vγ and Vδ classes of human TCR V-regions, implying a specific and an important role of γδ-cells in the maintenance of the immune homeostasis. Whether this implication represents a rule associated with self-tolerance, will be investigated by future analyses.     

Effect of epitalon on the lifespan increase in Drosophila melanogaster

The geroprotector activity of epitalon, a synthetic tetrapeptide Ala–Glu–Asp–Gly, was studied on the Drosophila melanogaster wild strain Canton-S. The substance was added to the culture medium only at the developmental stage (from egg to larva). Epitalon significantly increased the lifespan (LS) of imagoes by 11–16% when applied at unprecedented low concentrations — from 0.001×10^–6 to 5×10^–6 wt.% of culture medium for males and from 0.01×10^–6 to 0.1×10^–6 wt.% of culture medium for females. The increase in LS did not depend on the substance dose. Effective concentrations of epitalon were 16 000–80 000 000 times lower than those of melatonin. The possible mechanisms of the antioxidant and regulatory effects of epitalon are discussed.     

A method for the determination of the adherence of granulocytes to microtitre plates

We report a new partially automated method for the measurement of the adherence of PMN in vitro. Adherence to a plastic surface was detected by measuring leukocyte alkaline phosphatase activity of the adherent cells, with a Titertek Multiscan system.

Using three different cellular concentrations (1 × 10⁶, 5 × 10⁵, 2.5 × 10⁵ PMN/ml) the response curve was linear to 45 min and adhesion was maximal by 30 min. The specificity of the reaction was acceptable as was the assay reproducibility (intra-assay coefficient of variation <8%; inter-assay coefficient of variation <11%).     

Permeability of amalgam restorative materials

The beneficial effect of fluoride-containing amalgam in preventing recurrent dental caries depends on the ability of the material to deliver fluoride (F). A two-chamber diffusion cell has been employed to monitor the diffusion of F in freshly prepared amalgam sections (membranes) as well as in amalgam sections stored in F solution for 15 d. The diffusion of ¹²⁵I was also monitored, as a reference. Five and ten successive measurements at 72 h intervals were made on the fresh and stored amalgam specimens, respectively. The average diffusion coefficient, D, of F and ¹²⁵I in fresh amalgam was 2.39 × 10⁻¹⁰ and 1.85 × 10⁻¹⁰ cm²/s, respectively. For stored amalgam, the average D of F during a 30 d experiment was 1.35 × 10⁻¹⁰ cm²/s. The average D of F in stored amalgam, during the first 15 d of the experiment, was 31 % less than in fresh amalgam (p < 0.01). A decline in the diffusion process was observed during the course of the experiments. During 15 d diffusion in fresh amalgam and 30 d diffusion in stored amalgam the cumulative diffused F were 0.79 and 0.88% of the F in the source. SEM findings revealed the deposition of corrosion products on amalgam stored for 3 months in 0.19% F solution.     

Trophic action of pharmacological substances with a guanidine group on mouse neuroblastoma cells and chick ganglionic neurons in culture

A series of substances (designated CTQ compounds) with a guanidine group have been synthesized and tested for their ability to promote neuronal survival and neurite outgrowth. Mouse neuroblastoma clonal cell lines grown in serum-containing medium for 10 days as well as primary cultures of embryonic chicken ganglion neurons grown in serum-free defined medium for 1 or 2 days have been used for the experiments. Among the various CTQ compounds (CTQ1–CTQ20) tested, only CTQ8 exerted positive neurotrophic effects on these peripheral neuronal cells. At a concentration of 10⁻⁴ M, CTQ8 enhanced neuritogenesis of neuroblastoma cells. However, the most striking influence of CTQ8 was its promoting effect (6- to 10-fold) on the survival of chicken ciliary and dorsal root ganglionic neurons at concentrations ranging from 10⁻³ M to 5×10⁻⁴ M.     

Corrosion analysis of NiCu and PdCo thermal seed alloys used as interstitial hyperthermia implants

Ferromagnetic materials with low Curie temperatures are being investigated for use as interstitial implants for fractionated hyperthermia treatment of prostatic disease. Previous investigations of the system have utilized alloys, such as NiCu, with inadequate corrosion resistance, requiring the use of catheters for removal of the implants following treatment or inert surface coatings which may interfere with thermal characteristics of the implants. We are evaluating a palladium—cobalt (PdCo) binary alloy which is very similar to high palladium alloys used in dentistry. Electrochemical corrosion tests and immersion tests at 37 °C for both NiCu and PdCo alloy samples in mammalian Ringer's solution were performed. Long-term corrosion rates are 5.8 × 10⁻⁵ μm per year (NiCu) and 7.7 × 10⁻⁸ μm per year (PdCo) from average immersion test results, indicating higher corrosion resistance of PdCo (P < 0.02); immersion corrosion rates were much lower than initial corrosion rates found electrochemically. Both alloys had significantly lower corrosion rates than standard surgical implant rates of 0.04 μm per year (P < 0.001 for both alloys). Scanning electron microscopy illustrates changes in the NiCu alloy surface due to pitting corrosion; no difference is observed for PdCo. The data indicate that the PdCo alloy may be suitable as a long-term implant for use in fractionated hyperthermia.     

Effects of TGF-β on Eosinophil Chemotaxis

Transforming growth factor-β (TGF-β), which can decrease the effects of interleukin (IL)-3, IL-5 and granulocyte–macrophage colony-stimulating factor (GM-CSF) on eosinophil viability, has been shown to be chemotactic for neutrophils. However, there is little information on its effects on eosinophil chemotaxis. Because TGF-β has recently been found in increased concentrations in asthmatic sputum, we investigated whether TGF-β could influence eosinophil migration and eosinophil viability. Purified eosinophils from normal donors were incubated with increasing concentrations of TGF-β. Chemotaxis was measured with a modified Boyden chamber technique. In addition, eosinophils were incubated for 96 h with either IL-3, IL-5 or GM-CSF (1 ng/ml) together with increasing concentrations of TGF-β. Eosinophil viability was then determined with propidium jodide and flowcytometry. Eosinophil chemotaxis was significantly increased in the presence of TGF-β in concentrations between 10⁻⁹ and 10⁻⁴ μg/ml. The optimal concentration of TGF-β in this assay was between 10⁻⁹ and 10⁻⁸ μg/ml. The chemotactic effect of TGF-β diminished when higher as well as lower concentrations (between 10⁻¹² and 10⁻³ μg/ml) were employed. In contrast, inhibition of eosinophil survival induced by IL-3, IL-5 and GM-CSF reached its maximum at concentrations of TGF-β between 10⁻⁴ and 10⁻³ μg/ml. From these data we conclude that TGF-β in low concentrations can induce eosinophil chemotaxis whereas higher concentrations reduce eosinophil survival mediated by IL-3, IL-5 and GM-CSF.     

Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system

Isolated hepatocytes in spheroid configuration exhibit a high degree of cell–cell contacts, which are important in the maintenance of viability and liver specific functions. In the absence of a vascular network, the cells in a large spheroid size experience mass transfer limitations of metabolites and oxygen in the core of aggregates. In this paper transport phenomena related to the diffusion and reaction of oxygen, glucose and lactate are mathematically described and experimentally verified for hepatocyte spheroids cultured in a rotating-wall polystyrene system (RWPS) not permeable for gases and in a rotating-wall membrane system (RWMS) with oxygen-permeable membrane. The concentration profiles of glucose, oxygen and lactate in the hepatocyte spheroids were estimated for different diameters of aggregates by solving the mass transfer equations for simultaneous diffusion and reaction, by finite element method. Simulation results evidenced that, for aggregates with size lower than 300 μm cultured in both RWPS and RWMS systems, the concentration profiles of glucose and lactate towards the core of spheroids (effective diffusion coefficients in the order of 10⁻¹¹ m²/s) are not significantly affected by the metabolic rate (c.a 10⁻⁶ μg/mm³/s for glucose and about one order of magnitude less for lactate). On the contrary, the transport of oxygen (diffusion coefficient: 3.4×10⁻¹⁰ m²/s, reaction rate: 1.5×10⁻⁵ μg/mm³/s) is critically affected by the size of the multicellular spheroids and significant gradients in oxygen concentration may develop in spheroids. Aggregates with a size greater than 200 μm suffer severe oxygen limitation in the most part of its size attaining the lowest partial pressure in the centre. The improved viability predicted by the model culturing hepatocyte spheroids in the RWMS, characterized by a higher O₂ permeability with respect to RWPS, was experimentally confirmed. The results demonstrated that the mathematical model used in this study represents a useful support to experimental procedures in order to obtain hepatocyte spheroids with optimal size.     

SLOS carrier frequency in Poland as determined by screening for Trp151X and Val326Leu DHCR7 mutations

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations in the DHCR7 gene. Previous studies estimated the prevalence of SLOS between 1 in 10,000 to 1 in 70,358 based on case frequency surveys. Although panethnic, SLOS appears to be most frequent in Central European populations (Czech Republic 1 in 10,000, Slovakia 1 in 15,000 – 1 in 20,000). In Polish individuals with SLOS two DHCR7 mutations, c.452G > A (p.Trp151X) and c.976G > T (p.Val326Leu), account for 65.2% of all observed DHCR7 mutations. We analyzed 2169 samples for the p.Trp151X mutation and 2087 for the p.Val326Leu mutation. The combined carrier frequency of these two mutations of was 2.40 ± 0.32%, yielding a calculated incidence of SLOS in Poland of 2.5 4 × 10⁻⁴–4.3 5 × 10⁻⁴ (1 in 2,300 to 1 in 3,937) placing SLOS among the most common recessive genetic disorders in Poland.     

A Two Step Purification of Recombinant Human Interleukin-1β Expressed in E. Coli

Recombinant human interleukin-1β has been expressed in high yield using E. coli with a cDNA clone obtained from SKhep1RNA. The rIL-1β is purified to apparent homogeneity using freeze-thaw extractions followed by hydrophobic interaction chromatography over phenyl Sepharose. The procedure can provide pure rIL-1β (up to 15 mg per liter of E. coli culture) without the use of denaturants and if desired, in the absence of column chromatographic steps. Purity is defined by the presence of a single band on 1-D polyacrylamide gels and a single spot on 2-D polyacrylamide gels. The purified protein exhibits a biological activity of 1 × 10⁷ units/mg in a fibroblast proliferation assay and is shown to cross-react with rabbit anti-human IL-1β sera.     

Lack of stimulatory effect of dienogest on the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by endothelial cell as compared with other synthetic progestins

Objectives: Monocyte adhesion to endothelial cells is an important initial event at the onset of atherosclerosis. It is partially mediated by the expression of adhesion molecules on the endothelial cell surface. While estrogens inhibit the development of atherosclerosis, the effect of co-administered progestin remains controversial. We examined the effect of progestins on cytokine-stimulated human umbilical venous endothelial cell (HUVEC) expression of adhesion molecules. Methods: In HUVECs, mRNA expression of progesterone receptors (PRs) and androgen receptors (AR) was determined by RT-PCR. HUVECs were stimulated by interleukin-1β (IL-1β) for 24 h with or without various steroids, and then the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was semiquantified by ELISA. Results: In all preparations of HUVECs used in this study, RT-PCR confirmed mRNA expression of both isoforms of PR, PR-A and PR-B, as well as AR. Addition of progesterone (10⁻¹⁰–10⁻⁷ M) or dienogest (DNG) (10⁻¹⁰–10⁻⁸ M) did not affect IL-1β-stimulated ICAM-1 or VCAM-1 expression. In contrast, medroxyprogesterone acetate, norethindrone acetate and levonorgestrel (10⁻¹⁰–10⁻⁸ M) dose-dependently increased cell adhesion molecules. The progestin-induced increase was blocked by the concomitant addition of mifepristone, a PR antagonist, but not by hydroxyflutamide, an AR antagonist, indicating that the progestin stimulation was mediated predominantly via PR. Conclusions: These results suggest that DNG, unlike other synthetic progestins, lacks stimulation of cell adhesion molecules. For the prevention of atherosclerosis, estrogen in combination with DNG may be a suitable regimen in hormone replacement therapy in postmenopausal women.     

An assay to quantitate the binding of leishmania amastigotes to macrophages

A leishmania amastigote radiobinding assay has been developed using organisms labeled with tritiated uracil. These labeled amastigotes resemble freshly isolated unlabeled amastigotes in metabolic activity, bouyant density, morphology, viability and their ability to transform into promastigotes. Organisms routinely incorporate between 5 × 10⁻³ and 3 × 10⁻² cpm per amastigote, which allows the detection of as little as 1 × 10⁴ amastigotes per assay well. This radiolabeling technique has been used to quantitate the attachment of amastigotes to macrophages adherent to either 13 mm coverslips or to 96 well plates. It can also be used to screen monoclonal antibodies to macrophage surface proteins involved in amastigote binding. Once incorporated, the label remains amastigote associated, even after intact organisms have been internalized by macrophages. It remains parasite associated until the organisms have been degraded by macrophages, at which time label is released into the supernatant. Thus, a small adaptation of the binding assay can be used to compare the intracellular survival of amastigotes in macrophages following various experimental manipulations. This amastigote radiolabeling assay, therefore, represents an important step toward determining the receptors on macrophages involved in amastigote recognition and can also be used to study the degradation of intracellular pathogens by macrophages.     

Inhibitoty Effects of Local Anesthetics on Migration, Extracellular Release of Lysosomal Enzyme, and Superoxide Anion Production in Human Polymorphonuclear Leukocytes

This study examined the effects of four typical local anesthetics, lidocaine, prilocaine, procaine and tetracaine, on the functioning of human polymorphonuclear leukocytes (PMN). PMN were stimulated by fMet-Leu-Phe (FMLP) or phorbol myristate acetate (PMA) to elicit chemotaxis, extracellular release of beta-glucuronidase (BGL) and superoxide anion (SOA) production. the four agents inhibited chemotaxis efficiently and in a concentration-dependent manner but had only weak effects on the release of BGL. the effect of tetracaine was strongest, followed by lidocaine, then prilocaine, whereas the effect of procaine was blunt. the 50% inhibitory concentrations (IC₅₀ in molarity) of the four local aesthetics for chemetaxis were as follows: tetracaine=4.1×10^-4, lidocaine=3.2×10^-3, prilocaine=3.6×10, procaine=4.9×10^-3, those for SOA production induced by FMLP were : tetraaine=3.1×10^-4, lidocine=5.9×10^-3, prilocaine=1.9×10^-2, procaine=1.2×10^-2, those for SOA production indced by PMA were : tetracaine=1.1×10^-3, lidocine=1.2×10^-2, prilocaine=1.5×10-2, procaine=2.5×10^-2, and those for rlease of BGL were : tetracaine=1.6×10^-, lidocaine=5.3×10^-3, prilocaine=2.8×10^-2, procaine=1.2×10^-1. the IC₅₀ seemed to relate to the anesthetic's chemical structures and their inhibitory properties on PMN functions, as lidocaine and prilocaine, which are aminoamide type anesthetics, preferentially inhibited chemotaxis, whereas tetracaine and procaine, aminoester type anesthetics, inhibited SOA production induced by FMLP. the results suggest that the inhibitory effects of local anesthetics on human PMN functions are also correlated with local anesthetic potency and vary according to differences in their chemical structures.     

Heterogeneity in terms of interleukin 4-dependent regulation of Fce receptor/CD23 expression on chronic B-lymphocytic leukemia cells

To discriminate the stages of maturation arrest of leukemic B cells, we have investigated the cell surface expression of FcεR_1l (H107 antigen) on leukemic B cells from 6 patients with chronic type B-lymphocytic leukemia(B-CLL) by a double staining method combined with cytoflorometry, and their production of soluble FceR_ll ⁺ by an ELISA technique. FceR_ll was expressed onμ⁺/δ⁻ cells of case 5 as well as on μ⁺/δ⁺cells of cases 1,2 and 4, but not on μ⁺/δ⁺cells in cases 3 and 6. The cultivation of leukemic cells with IL-4 not only increased the percentage of FceR_ll⁺cells but also enhanced the production of soluble Fcer_llin most cases. However, IL-4 had no effects on μ⁺/δ⁻/Fcεr_ll⁺ cells of cases 5, which appeared to correspond to a rather late     

Biomechanics of the saphenous artery and vein in spontaneous hypertension in rats

Incremental compliance and distensibility of certain muscular arteries were recently reported to be normal or slightly increased in hypertension at the same pressure levels. In this work biomechanical properties of isolated perfused and superfused veins and large muscular arteries from saphenous bed from male spontaneously hypertensive (SHR) and Wistar–Kyoto (WKY) rats were compared in vitro. Outer diameter of cylindrical vessel segments was measured and intraluminal pressure (IP) was changed cyclically. We found larger contractile response to methoxamine (1.06×10⁻⁵ mol/l) in SHR arteries compared to WKY (active strain e.g. at 100 mmHg IP: 7.12±4.1 vs 0.35±0.46%). Resting incremental distensibility was higher (e.g. 100 mmHg IP: 3.4±0.4×10⁻⁶ vs 1.2±0.3×10⁻⁷ m²/N), elastic modulus lower (e.g. 100 mmHg IP: 3.7±0.6×10⁵ vs 27±7.6×10⁵ N/m²) in the arteries from SHR in pressure range of 60–110 mmHg. After papaverine administration (2.8×10⁻⁴ mol/l) the artery became more rigid, thus the increased incremental elasticity of SHR artery might be due to the enhanced smooth muscle tone. However, compared at in vivo pressure levels the differences were negligible suggesting a shift in the elastic parameters toward the higher operation pressures. Saphenous vein of SHR had larger diameter, than that of WKY, while in the wall thicknesses no difference were found (therefore external radius-wall thickness ratio was larger, e.g. at 6 mmHg IP: 15.9±3.0 vs 8.1±0.7). Consequently, lumen capacity of the vein was also higher in SHR, however, elastic parameters did not exhibit significant differences. We conclude that pressure-distensibility curve of muscular type arteries like SHR saphenous artery is shifted to higher pressure levels compared with that of normotensive controls. This shift is due to the enhanced smooth muscle contractility. The unchanged elasticity of veins suggests that the arterial deformations in SHR are not primary but secondary alterations.     

Influence of Salbutamol and Isoproterenol on the Production of TNF and Reactive Oxygen Species by Bovine Alveolar Macrophs and Calcitriol Differentiated HL-60 Cells

The influence of the β-adrenergic agonists Salbutamol and Isoproterenol on the release of reactive oxyen species and Tumor Necrosis Factor (TNF) was tested with bovine alveolar macrophages and HL-60 cells differentiated to macrophages by calcitriol. The production of reactive oxygen species was analyzed by a microplate assay using dichlorofluorescein-diacetate. It could be shown that this method almost exclusively measures superoxide anions. TNF was determined by a bioassay with WEHI cells. The superoxide anion production was stimulated by Zymosan, the TNF release by LPS. By incubation with 5×10^-6 and 5×10^-7 M Salbutamol or 5×10^-7 and 5×10^-8 m lsoproterenol prior to the stimulation, the production of superoxide anions as well as of TNF was inhibited to a significant deree. The inhibitory effects of the adrenergic agonists were completely or at least partially inhibited by the respective antagonists, ICI 118.551 and Propanolol, respectively.     

Acquisition of lipoproteins in the procyclic form of Trypanosoma brucei

The procyclic form of Trypanosoma brucei binds and internalizes bovine high density lipoprotein (HDL) particles in a saturable process; the binding and uptake of ¹²⁵I-labeled HDL are inhibited by excess unlabeled HDL. We calculated that each procyclic trypanosome exposes ≈1.0×10⁶ binding sites for bovine HDL, with an equilibrium dissociation constant (K_d) of ≈1.26×10⁻⁷ M. Uptake of HDL particles does not occur at 4°C. At 28°C, a significant amount of the internalized HDL particles were efficiently degraded through a process that is sensitive to the presence of 50 μM chloroquine. These results suggested that the uptake of HDL particles in procyclic T. brucei may occur via receptor mediated endocytosis, leading to proteolytic degradation of the particles in an acidic and endocytic compartment.