Refsum disease diagnostic marker phytanic acid alters the physical state of membrane proteins of liver mitochondria.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), a branched chain fatty acid accumulating in Refsum disease to high levels throughout the body, induces uncoupling of rat liver mitochondria similar to non-branched fatty acids (e.g. palmitic acid), but the contribution of the ADP/ATP carrier or the aspartate/glutamate carrier in phytanic acid-induced uncoupling is of minor importance. Possible deleterious effects of phytanic acid on membrane-linked energy coupling processes were studied by ESR spectroscopy using rat liver mitochondria and a membrane preparation labeled with the lipid-specific spin probe 5-doxylstearic acid (5-DSA) or the protein-specific spin probe MAL-TEMPO (4-maleimido-2,2,6, 6-tetramethyl-piperidine-1-oxyl). The effects of phytanic acid on phospholipid molecular dynamics and on the physical state of membrane proteins were quantified by estimation of the order parameter or the ratio of the amplitudes of the weakly to strongly immobilized MAL-TEMPO binding sites (W/S ratio), respectively. It was found, that phytanic acid (1) increased the mobility of phospholipid molecules (indicated by a decrease in the order parameter) and (2) altered the conformational state and/or the segmental mobility of membrane proteins (indicated by a drastic decrease in the W/S ratio). Unsaturated fatty acids with multiple cis-double bonds (e.g. linolenic or arachidonic acid), but not non-branched FFA (ranging from chain length C10:0 to C18:0), also decrease the W/S ratio. It is hypothesized that the interaction of phytanic acid with transmembrane proteins might stimulate the proton permeability through the mitochondrial inner membrane according to a mechanism, different to a protein-supported fatty acid cycling.

Protection of mitochondrial integrity from oxidative stress by the triaminopyridine derivative flupirtine.

The suitability of the triaminopyridine derivative flupirtine, an analgesic drug with antioxidative property [Gassen, M., Pergande, G. and Youdim, M.B.H. (1998) Biochem. Pharmacol. 56, 1323-1329], for the preservation of mitochondrial integrity from oxidative stress-induced damage was studied. Rat liver mitochondria were exposed to strong oxidative stress as generated by Fe(2+) plus ascorbate. Peroxidation damage of membrane lipids was followed by the measurement of thiobarbituric acid reactive substances. Protein oxidation was estimated by electron spin resonance spectroscopy, after labeling of the 'peroxidized' mitochondria with 4-maleimido-2, 2,6,6-tetramethylpiperidine-1-oxyl. We found that (i) low concentrations of flupirtine (10 microM) protect lipids and also proteins (with lesser efficiency) from attacks of reactive oxygen species; (ii) flupirtine remarkably delayed the decline of complex mitochondrial functions, such as the respiratory control or the Ca(2+) retention capacity of mitochondria, under oxidative stress; and (iii) the ADP/ATP antiporter (ANT), a main component of the oxidative phosphorylation machinery as well as a core component of the permeability transition pore complex, seems to be a membrane protein particularly protected by flupirtine. In conclusion, the preservation of the Ca(2+) buffer capacity of mitochondria and of the ANT activity against oxidative stress supports an antiapoptotic application of flupirtine.

Effect of granulocyte colony-stimulating factor treatment on ex vivo neutrophil functions in nonneutropenic surgical intensive care patients.

Granulocyte colony-stimulating factor (G-CSF) preferentially stimulates growth and differentiation of neutrophil precursors and activates neutrophil functions. The aim of the present study was to investigate the functional response of the neutrophil to exogenous recombinant human G-CSF (rHuG-CSF) in nonneutropenic patients. In 30 surgical intensive care unit patients with severely impaired wound healing, leukocyte differential count, plasma G-CSF level, and a broad spectrum of neutrophil functions were monitored before (day 0), throughout (days 1 and 5), and at days 1 and 5 after stopping G-CSF treatment. G-CSF application resulted in a 3.5-fold increase in peripheral blood granulocyte count at day 5 of treatment. The mean plasma G-CSF level rose from 48 to a maximum of 2314 pg/ml at day 1 of G-CSF therapy. Neutrophil chemotaxis and stimulated lysozyme release were decreased throughout G-CSF treatment, whereas respiratory burst activity, phagocytic activity, and intracellular calcium concentration were enhanced by G-CSF. Neutrophil membrane depolarization remained unaffected. The increased count and activation state of neutrophils were associated with clinical improvement in most of these patients. Thus, G-CSF may be a useful adjuvant treatment for nonneutropenic patients with severely impaired wound healing.

[Detection and differentiation of autoantibodies to extractable nuclear antigens with immunoenzyme tests]. / Untersuchungen zum Nachweis und zur Differenzierung von Autoantikörpern gegen extrahierbare Kernantigene mit Enzymimmuntesten.

Nuclear antigens were extracted from calf thymus with phosphate-buffered saline The 60-80% ammonium sulfate fraction (ASF) of thymus extract contained SS-B-, the 30-60% ASF Sm- and U1-RNP-, and the chromatographically purified 30-60% ASF only Sm antigen. With these fractions enzyme immunoassays were developed for quantitative determination of Sm-, U1-RNP-, and SS-B autoantibodies. Out of 144 sera with antinuclear antibodies 85% were positive in the enzyme immunoassays, 41% in immunofluorescence tests on liver sections (12% speckled, 29% homogeneous immunofluorescence pattern), and 10% in Ouchterlony tests. 26% of the sera were positive in enzyme immunoassays and immunofluorescence tests. All antibody specificities detected by immunodiffusion could be confirmed by enzyme immunoassay. The enzyme immunoassay is far more sensitive then immunodiffusion and in contrast to immunofluorescence allows antibody specificities to be determined. Enzyme immunoassays are recommended for the diagnosis of rheumatic diseases.

[Determination of immune complexes in the serum of pregnant patients and puerperal females with EPH gestosis]. / Bestimmung von Immunkomplexen im Serum von Schwangeren und Wöchnerinnen mit EPH-Gestose.

We analyzed the blood sera from 100 patients with EPH gestosis and from 50 women with normal pregnancy to explore the immunological reactions during gestosis. The sera of cord blood were included in the investigations. We determined the circulating immune complexes by conglutinin and C1q solid phase radioimmunoassay and by polyethyleneglycol precipitation. It has been established by conglutinin RIA that during pregnancy the women with gestosis have significant higher concentrations of immune complexes than the control group. During puerperium and in the cord blood the values are significantly lower than during pregnancy in both groups. The estimations of immune complexes by C1q RIA have shown that the differences between the two groups are insignificant and the lowest values are in the cord blood. The concentrations of immune complexes estimated by PEG precipitation were not significantly different between the two groups. It is concluded that immune complexes are in connection with the pathogenesis of preeclampsia.

Protection against UVA damage and effects on neutrophil-derived reactive oxygen species by beta-carotene.

OBJECTIVE: Phagocyte-derived reactive oxygen species (ROS) are involved in microbicidal activities as well as in tissue damage at sites of inflammation. Carotenoids play an important function in protecting cells from oxidant damage. We investigated the in vitro and in vivo effect of 13-cis and 9-cis-beta-carotene on human neutrophils. METHODS: Neutrophils from healthy donors in the presence of 0.25 mumol/L-1 mumol/l beta-carotene and from subjects under beta-carotene supplementation and UVA or UVA/B exposure were stimulated by opsonized zymosan and the generation of ROS was measured by electron spin resonance spectroscopy. RESULTS: Our in vitro results show different effects of the two isomers on stimulated neutrophils. 9-cis-beta-carotene did not produce any change, whereas 13-cis-beta-carotene significantly and concentration-dependent inhibited the ROS generation by stimulated neutrophils. Further, in a controlled study, we were able to demonstrate an in vivo protective effect of beta-carotene on neutrophils against UVA damage by beta-carotene supplemented subjects.

Effects of retinoids on the generation of neutrophil-derived reactive oxygen species studied by EPR spin trapping techniques.

OBJECTIVE: To study the potential efficacy of retinoids on granulocytes with regard to their role in inflammatory dermatoses. METHODS: We investigated the in vitro effect of acitretin and isotretinoin on the generation of reactive oxygen species (ROS) by stimulated human neutrophils using EPR spin trapping techniques. RESULTS: The effects of the two retinoids on ROS generation by neutrophils were different. Acitretin increased the generation of hydroxyl radicals, whereas isotretinoin showed an antioxidant activity against the superoxide anion. CONCLUSIONS: Our study demonstrates retinoid type-dependent effects on ROS production by stimulated neutrophils.

[Determination of thyroglobulin autoantibodies using enzyme immunoassay, radioimmunoassay and passive hemagglutination]. / Untersuchungen zum Nachweis von Thyreoglobulin-autoantikörpern mit Enzymimmuntest, Radioimmuntest und passiver Hämagglutination.

A solid-phase enzyme immunoassay, a radioimmunoassay and the passive hemagglutination were tested for the determination of antithyroglobulin autoantibodies. The antibody-titres detected with these methods showed a good correlation. The sensitivities of the solid-phase immunoassays are four times higher than the sensitivity of the passive hemagglutination. For the diagnosis of thyroid autoimmune diseases the enzyme immunoassay.

[Characterization of treponemal axial filament antigens using the Western blot]. / Charakterisierung der Treponemen-Axialfilamentantigene im Westernblot.

Analysis by SDS-PAGE of axial filaments of cultivable treponemes showed 3 major bands: a 33/34 kD doublet and a 37 kD polypeptide. In sera from patients with various stages of syphilis, from biological false positive reactors, and from negative controls the most consistent reaction was detected against the doublet, but only sera from patients with primary or secondary syphilis reacted with the 37 kD axial filament polypeptides. About 6 weeks after antibiotic treatment sera had lost their reactivity with 37 kD antigens in agreement with decreasing antibodies in VDRL-test. Because of their recognition early in infection 37 kD-proteins are postulated to use in serodiagnostic assays, but our data emphasize also the need for use further antigens in tests for diagnosis in later stages of syphilis.

[Detection of circulating immune complexes in patients with EPH gestosis or intrauterine fetal growth retardation]. / Nachweis von zirkulierenden Immunkomplexen bei Patientinnen mit EPH-Gestose oder intrauteriner fetaler Retardierung.

Sera from 19 patients with EPH gestosis, 22 patients with hypotrophic newborns, and a control group of ten women with normal pregnancy and fetuses were studied for the presence of circulating immune complexes. Both the C1q solid-phase radioimmunoassay and the polyethylene glycol precipitation test revealed significantly higher levels of circulating immune complexes in both groups of patients.