Efficacy of permethrin-impregnated bed nets on malaria control in a hyperendemic area in Irian Jaya, Indonesia III. Antibodies to circumsporozoite protein and ring-infected erythrocyte surface antigen.

A two years intervention study was carried out using permethrin impregnated bed nets in a hyperendemic area, in Irian Jaya, Indonesia. To assess the influence of this intervention on natural immunity, concurrent immunological studies to determine levels of antibodies to the circumsporozoite (CS) and ring-infected erythrocyte surface antigen (RESA) proteins were conducted. Prevalence and titers of immunoglobulins (Ig)G and IgG subclasses were periodically measured in 138 individuals (30 children under the age of ten and 108 villagers ten years old and older). In the younger group, seropositivity of total IgG against CS fluctuated according to the parasite infection rates; however, IgG seropositive reaction against RESA gradually increased. In the older age group, seropositivity of both kinds of antibodies was stable during the whole study period. Nevertheless, the geometric mean titers of total IgG against CS and RESA were significantly reduced in this latter group in individuals who contained these antibodies before and after intervention. The geometric mean titer of IgG3 subclass against RESA was decreased at a highly significant level (p = 0.0005), and that of IgG4 against the same antigen was also decreased although to a lesser extent (p = 0.02).

Iron status and oxidative stress in beta-thalassemia patients in Jakarta.

A study on thalassemia intermedia and major patients in Jakarta was initiated to obtain a comprehensive picture of metabolic dysregulation, iron overload, oxidative stress, and cell damage. Data are presented from a group of 14 transfusion-dependent patients in an age range of 11-25 years (T) and another group of 9 frequently transfused (for at least 15 years) patients aged 17-30 years (L). A third group comprised 6 patients (aged 7 to 14 years) who had not yet obtained transfusions (N). The 21 controls (C) were voluntary students without diagnosis or clinical signs of thalassemia up to 30 years of age. The study was approved by the Ethical Clearance Board of the Medical Faculty and all blood samples from controls and patients were obtained on fully informed consent. Levels of antioxidants (vitamins A, C, E and beta-carotene) and reactive thiols are considerably decreased in transfused patients, whereas signs of iron overload and cell damage are increased (serum iron, ferritin, transferrin saturation, SGOT, SGPT, gamma-GT, bilirubin). Results can be summarized that non-transfused thalassemia intermedia patients exert slight signs of oxidative stress, and increased hemoglobin degradation but no significant indication of tissue or cell damage. This picture differs considerably from transfusion-dependent thalassemia major patients: highly significant decrease in antioxidants and thiols and tremendous iron overload and cell damage. The picture is even worsened in long-term transfused patients. Iron chelation after transfusion is not sufficient in Indonesia, because it is normally (with few exceptions) applied only once together with transfusion. Hence, one major reason of the bad condition of transfusion-dependent thalassemia patients in Indonesia appears to be frequent transfusions (on the average one per month) and insufficient chelation of one treatment per month together with transfusion.

Simple and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis.

A rapid and sensitive PCR assay for the detection of Candida albicans DNA in serum was established. DNA from human serum samples was purified using the QIAamp blood kit, which proved to be a fast and simple method for isolating minute amounts of Candida DNA from clinical specimens for diagnosis of invasive candidiasis. Universal primer sequences used in the PCR assay are derived from the internal transcribed spacer rRNA gene of fungi, whereas the biotinylated hybridization probe used in a DNA enzyme immunoassay (DEIA) binds specifically to C. albicans DNA. The sensitivity of this PCR-DEIA method is very high; the detection limit for genomic Candida DNA is one C. albicans genome per assay. Blood from uninfected and infected persons, ranging from healthy volunteers, patients with mucocutaneous infections, and patients at risk to develop a systemic Candida infection to patients with an established systemic candidiasis, was analyzed for the presence of C. albicans to diagnose fungal infection. Candida DNA could not be detected in sera of 16 culture-negative controls and from 11 nonsystemic candidal infections by PCR or DEIA. Blood cultures from patients at risk were all negative for Candida, whereas all blood cultures from systemic candidiasis patients were positive. However, Candida DNA could be detected by PCR and DEIA in the serum from three out of nine patients who were at risk for a systemic infection and in the serum of all seven patients who had already developed an invasive Candida infection. PCR is more sensitive than blood culture, since some of the patients at risk for invasive yeast infection, whose blood cultures were all negative for Candida, tested positive in the PCR amplification. These results indicate the potential value of PCR for detecting C. albicans in serum samples and for identifying patients at risk for invasive candidiasis.

Lipoic acid reduces ischemia-reperfusion injury in animal models.

Hypoxia and reoxygenation were studied in rat hearts and ischemia and reperfusion in rat hindlimbs. Free radicals are known to be generated through these events and to propagate complications. In order to reduce hypoxic/ischemic and especially reoxygenation/reperfusion injury the (re)perfusion conditions were ameliorated including the treatment with antioxidants (lipoate or dihydrolipoate). In isolated working rat hearts cardiac and mitochondrial parameters are impaired during hypoxia and partially recover in reoxygenation. Dihydrolipoate, if added into the perfusion buffer at 0.3 microM concentration, keeps the pH higher (7. 15) during hypoxia as compared to controls (6.98). The compound accelerates the recovery of the aortic flow and stabilizes it during reoxygenation. With dihydrolipoate, ATPase activity is reduced, ATP synthesis is increased and phosphocreatine contents are higher than in controls. Creatine kinase activity is maintained during reoxygenation in the dihydrolipoate series. Isolated rat hindlimbs were stored for 4 h in a moist chamber at 18 degrees C. Controls were perfused for 30 min with a modified Krebs-Henseleit buffer at 60 mmHg followed by 30 min Krebs-Henseleit perfusion at 100 mmHg. The dihydrolipoate group contained 8.3 microM in the modified reperfusate (controlled reperfusion). With dihydrolipoate, recovery of the contractile function was 49% (vs. 34% in controls) and muscle flexibility was maintained whereas it decreased by 15% in the controls. Release of creatine kinase was significantly lower with dihydrolipoate treatment. Dihydrolipoate effectively reduces reoxygenation injury in isolated working rat hearts. Controlled reperfusion, including lipoate, prevents reperfusion syndrome after extended ischemia in exarticulated rat hindlimbs and in an in vivo pig hindlimbs model.

Benefits of vitamin E supplementation to Norplant users--in vitro and in vivo studies.

Norplant subcutaneous implantation is a contraceptive method used in Indonesia. Endometrial bleeding is one major reason to discontinue the use of Norplant. Angiogenic response in the endometrium of Norplant users was found to be lower than in women with normal menstrual cycle. This disturbance in the angiogenic process may be caused by an imbalance of pro- and antioxidant processes in the endometrium of Norplant users. The aim of this study is to investigate the effect of vitamin E on the endometrial angiogenic activity and to assess the efficacy of vitamin E supplementation in treating endometrial bleeding in Norplant users. Subjects for this study were selected from Norplant users with an exposure of at least 3 months, with endometrial bleeding and recruited on the basis of fully informed consent. TBA reaction was used to measure degradation products of lipid peroxidation. The endometrial angiogenic response was assayed according to Folkman et al. (Folkman et al., 1989. Nature 239, 58-61). Samples from endometrial biopsies were incubated in vitro with vitamin E or placebo before angiogenic measurement. For in vivo supplementation, vitamin E 200 mg/day, or placebo for 10 days/month were given to the subjects with double blind randomisation. The results showed that the blood levels of TBA-reactive substances were significantly higher in Norplant users than in controls. In the endometrium from Norplant users with bleeding problems, in vitro supplementation of vitamin E resulted in a significantly higher angiogenic score than placebo. Although a highly significant reduction of bleeding days in both groups, vitamin E and placebo, was seen during the 2 months of the study, the number of bleeding days was significantly lower in women treated with vitamin E than with placebo.

Monomolecular organization of the main tetraether lipid from Thermoplasma acidophilum at the water-air interface.

The monomolecular organization of the main tetraether phospholipid from the archaeon Thermoplasma acidophilum was studied by means of a Langmuir film balance integrated into a fluorescence microscope. After transfer to solid surfaces at different pressures the films were further investigated by ellipsometry, small angle X-ray scattering and atomic force microscopy. In order to complete former results about the main tetraether phospholipid of T. acidophilum [Strobl, C., Six, L., Heckmann, K., Henkel, B., Ring, K., 1985. Z. Naturforsch. 40c, 219-222], the thickness and the two-dimensional organization of the monomolecular films were investigated. Two mean heights values were determined, one of 1.5-1.8 nm and another one of 4-5 nm, indicative for two different molecular arrangements. The former one is interpreted as a 'horseshoe' organization with two polar endings in the aqueous subphase, whereas the latter appears to represent the upright population of molecules with one polar end in the subphase and the other one in the air. In freshly spread and compressed films small domains of the upright lipid population are initially observed, which enlarge with increasing pressure. These domains are no longer existent after 12 h of spreading without compression.

Lipoate ameliorates ischemia-reperfusion in animal models.

Ischemia and reperfusion were studied in isolated working rat hearts and in exarticulated rat hind limbs. Free radicals are known to be generated in ischemia/reperfusion and to propagate complications. To reduce reperfusion injury, conditions were ameliorated including the treatment with antioxidants, lipoate or dihydrolipoate. In isolated working rat hearts, cardiac and mitochondrial parameters are impaired during hypoxia and partially recover in reperfusion. Dihydrolipoate, if added into the perfusion buffer at 0.3 microM concentration, keeps the pH higher (7.15) during hypoxia, as compared to controls (6.98). This compound accelerates and stabilizes the recovery of the aortic flow. With dihydrolipoate, ATP synthesis is increased, ATPase activity (ATP hydrolysis) reduced, intracellular creatine kinase activity maintained and thus phosphocreatine contents are higher than in controls. For exarticulated rat hind limbs, the dihydrolipoate group contained 8.3 microM in the modified reperfusate. Recovery of the contractile function was 49% vs. 34% in controls and muscle flexibility was maintained whereas it decreased by 15% in the controls. Release of creatine kinase from cells was significantly lower with dihydrolipoate. Lipoate/dihydrolipoate effectively reduced reperfusion injury in isolated working rat hearts and in exarticulated rat hind limbs after extended ischemia. Finally, the compound was successfully applied in an in vivo pig hind limb model.

Efficacy of permethrin-impregnated bed nets on malaria control in a hyperendemic area in Irian Jaya, Indonesia: influence of seasonal rainfall fluctuations.

A malaria intervention study was carried out using permethrin impregnated bed nets in the south-central part of Irian Jaya with perennial transmission, from April 1993 to April 1995. Malariometric surveys were carried out periodically for parasite prevalence by species and for spleen rates. Prior to intervention, the percentage of Plasmodium falciparum infected inhabitants was significantly higher in Hiripau, where permethrin-impregnated bed nets were used during the study, than in the placebo-treated control village, Kaugapu. After two years of intervention the situation was reversed and figures higher in the control village (RR 0.19, 95% CI 0.10-0.36, p < 0.0001). Similarly, P. vivax infection rates, 12.4% in Hiripau vs 5.7% in Kaugapu in April 1993. were reversed in April 1995 (3.6% in Hiripau and 11.3% in Kaugapu, p < 0.001). In the treated village, pre-control hyperendemicity was reduced to a low mesoendemic level (spleen rate 12.5%) during two years of intervention, whereas the level was mesoendemic (spleen rate 35.2%) in the control village. Impregnated bed nets were found an effective intervention both in moderate (April 1993 through April 1994, 1,626 mm rainfall) and high (April 1994 through April 1995/1995, 3,321 mm) transmission seasons.

Efficacy of permethrin-impregnated bed nets on malaria control in a hyperendemic area in Irian Jaya, Indonesia: differentiation between two age groups.

A malaria intervention trial was conducted for two years to evaluate the efficacy of permethrin-impregnated bed nets in reducing malaria infection and splenomegaly in two different age groups, ie below and over age of ten, in a hyperendemic area in Irian Jaya, Indonesia. Permethrin-impregnated or placebo-treated bed nets were provided to a treated and a control village, respectively. Immediately after periods with moderate rainfall in the first year, treated bed nets decreased P. falciparum and P. vivax density in the blood of children <10 years (group 1) but did not reduce the percentage of infection with either species. Children >10 and adults (group 2) showed significant reduction only in P. falciparum infection rates and density, whereas P. vivax was not influenced. After an excessive rainfall season in the second year, the risk for P. falciparum infections in both age groups using treated nets was less than half of that in the control village. P. vivax infection rates were significantly lower in the treated village at the beginning of and after these heavy rainfalls. In the treated village, spleen enlargement was markedly reduced in the younger age group during the second year.

Influence of selegiline and lipoic acid on the life expectancy of immunosuppressed mice.

Ten groups of 14 immunosuppressed NMRI-mice (nu/nu) were raised and kept under germ-reduced conditions. The control animals were fed a germ-reduced diet, nine other groups received the same diet with selegiline (CAS 14611-51-9, Deprenyl) or lipoic acid (thioctic acid, CAS 62-46-4) admixed at various amounts. The 50% survival rate, the total life span of each group and the areas under the curves were determined to evaluate life expectancy as compared to the controls. The racemate of lipoic acid at high dosage (350 mg/kg body weight) reduced the life span significantly. The S(-)-enantiomer of lipoic acid (75 mg/kg body weight) increased the 50% survival rate, whereas the physiologic R(+)-enantiomer (9 mg/kg body weight) expanded the total life span of its group. Alteration of only one out of three parameters was not considered significant. All other groups except for one did not differ from controls: only animals which obtained 75 micrograms selegiline per kg of body weight and per day exerted increased life expectancies by all three parameters. This group exhibited also in statistical evaluation a significantly (p < 0.05) prolongated survival time up to about 200% as compared to the control animals.

Ursodeoxycholate stabilizes phospholipid-rich membranes and mimics the effect of cholesterol: investigations on large unilamellar vesicles.

Ursodeoxycholate is used to treat primary biliary cirrhosis and is incorporated into hepatocyte plasma membranes. Its steroid nucleus binds to the apolar domain of the membrane, in a similar position to cholesterol. Therefore the question arises whether ursodeoxycholate has a similar effect on membrane structure and stability as cholesterol. Using differential scanning calorimetry the thermotropic behavior of egg phosphatidylcholine and dimyristoylphosphatidylcholine were studied after incubation with cholesterol or ursodeoxycholate. Large unilamellar vesicles were prepared with cholesterol contents of 0-50%. Following incubation of these vesicles with different amounts of ursodeoxycholate, vesicle stability in a gravitational field was investigated by measuring the phospholipid and cholesterol release. Vesicle size was studied by laser light scattering after incubation with cheno- and ursodeoxycholate, and the release of entrapped carboxyfluorescein was measured by means of fluorescence spectroscopy. Increasing cholesterol diminished the enthalpy of the phase transition in the membrane. Ursodeoxycholate decreased the enthalpy of the phase transition at even lower concentrations. Lipid release from vesicles in a high gravitational field diminished with increasing cholesterol content of the vesicles. Ursodeoxycholate had a comparable effect, which increased as the cholesterol content of the vesicles was decreased. Chenodeoxycholate damaged vesicles, whereas ursodeoxycholate did not. Cholesterol and ursodeoxycholate (below its critical micellar concentration) decreased the carboxyfluorescein release from vesicles induced by chenodeoxycholate. Thus like cholesterol, ursodeoxycholate is incorporated into phospholipid model membranes and reduces the change in enthalpy of the gel to liquid-crystalline phase transition. Like cholesterol ursodeoxycholate also maintains membrane stability and prevents membrane damage induced by mechanical and chemical stress.

Spin trapping study of antioxidant properties of neopterin and 7,8-dihydroneopterin.

Previously, it has been shown that pteridine derivatives are capable of modulating the action of free radicals and both prooxidant and antioxidant properties have been described. However, the mechanism of manifestation of these properties is still unclear. We studied the radical scavenging properties of 7,8-dihydroneopterin and neopterin using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). It was found that dihydroneopterin acts generally as a radical scavenger. In the presence of dihydroneopterin the ESR signal was reduced by 30 to 90% compared to the control signal. The rate constants for the reactions of 7,8-dihydroneopterin with superoxide (10(3) M(-1) s(-1)) and peroxyl radicals (10(7) M(-1) s(-1)) were determined. Neopterin in contrast showed no reduction of the ESR signal except with superoxide radicals produced by xanthine oxidase. However, this effect was shown to be due to an inhibition of enzyme rather than to radical scavenging. Our results provide a basis for understanding previous observations of radical scavenger activity of 7,8-dihydroneopterin.

Oxidative stress and antioxidant status in beta-thalassemia major: iron overload and depletion of lipid-soluble antioxidants.

Because of continuous blood transfusions, thalassemia patients are subjected to peroxidative tissue injury by the secondary iron overload. In accordance, analysis of serum from 42 beta-thalassemia patients, aged 4 to 40 years, showed that the mean concentrations of conjugated diene lipid hydroperoxides (CD), lipoperoxides evaluated as malondialdehyde/ thiobarbituric acid (MDA/TBA) adducts, and protein carbonyls increased about twofold with respect to control. Ferritin levels were positively correlated with the amount of MDA (r = .41; P = .007) and showed a positive trend with CD (r = .31; P = .07) and protein carbonyls (r = .35; P = .054), as further evidence of the deleterious effects of high tissue iron levels. Marked changes in the antioxidant pattern were also observed in all patients. Evidence is presented of a net drop in the concentration of ascorbate (-44%), vitamin E (-42%), vitamin A(-44%), beta-carotene (-29%), and lycopene (-67%). On the other hand, an increase of uric acid and bilirubin was observed, whereas serum albumin and glutathione were in the normal range in all patients. As a result, the total serum antioxidant potential, measured as trolox equivalent antioxidant capacity appeared significantly decreased by 14%. Serum levels of vitamin E were inversely correlated with ferritin (r = -.45; P = .003), suggesting a major consumption of this antioxidant under iron overload. Nontransferrin bound iron (NTBI) was in the range 4.5 to 54.8 micrograms/dL (mean, 21.8 +/- 13.9). Although NTBI had a positive trend with ferritin (r = .37, P = .03), no clear correlation was found with either MDA or vitamin E. A mild to severe hepatic damage, as assessed by serum transaminases, was shown in 24 of 42 patients. Serum levels of vitamin E (r = -.49, P = .015), vitamin A (r = -.48, P = .016) and lycopene (r = -.47, P = .020), were inversely correlated with the levels of transminases. On the other hand, lipid-soluble antioxidants in thalassemia patients were depleted to the same extent in hepatitis C virus (HCV)-infected (31 subjects) and in HCV-uninfected (10 subjects), while in the normal range in serum from 30 nonthalassemic patients with HCV-related chronic hepatitis. These results point out that the iron-induced liver damage in thalassemia may play a major role in the depletion of lipid-soluble antioxidants. The variations of the parameters evaluated in the present study were not correlated with the age of the patients. Our results suggest that the measurement of peroxidation products, matched with evaluation of antioxidants, may be a simple measure of iron toxicity in thalessemia, in addition to the conventional indices of iron status.

Interaction of glucose and metformin with isolated red cell membrane.

Isolated human erythrocyte membranes (red blood cell (RBC) ghosts) were incubated with glucose at 5, 10, 20 and 100 mmol/l concentrations, with insulin (0.01 to 200 mU/l) and metformin (CAS 657-24-9) 0.5 up to 50.0 mumol/l). Binding studies with 14C-glucose and subsequent gel electrophoresis revealed 60% of the radioactivity around ban 4.2-4.5 at 5 mmol/l, whereas a random distribution of radioactivity over all protein bands of the RBC membrane was found at 20 mmol/l concentration after incubation for 30 min or 48 h. Metformin does not bind covalently to RBC membranes, however, after photochemical linkage of 14C-metformin via the aminoreactive linker azidophenylglyoxal the highest radioactivity (21%) was counted in the range of band 4.2-4.5. In parallel with an increase of order parameters of 5-doxyl-stearic acid the thiol status of the membranes decreases as determined by monobromobimane fluorescence. 20 and 100 mmol/l concentrations of glucose decrease the reactivity of membrane thiols towards bromobimane significantly to 73 and 62% of the controls. Concomitantly, membrane fluidity at polar sites is diminished as measured by order parameters of spin label 5-doxyl stearic acid. In RBC membranes pretreated with 20 mmol/l glucose the decreased fluorescence is significantly raised again by insulin and metformin. This effect is even more pronounced, if insulin and metformin are incubated together. Reaction of membrane thiols with a maleimido spin label detects modification in the ratio of mobile and immobilized spin label populations in the electron paramagnetic resonance signal under the above conditions, indicative of conformational changes of membrane proteins.

Impact of oxidative stress on human cytomegalovirus replication and on cytokine-mediated stimulation of endothelial cells.

Transplantation-related pathogenic factors such as ischemia or allograft-directed inflammation are associated with oxidative changes that might lead to cellular oxidative stress. The aim of this study was to investigate the impact of oxidative stress on: (1) CMV replication in cultured human endothelial cells and (2) the stimulation of endothelial cells by proinfiammatory cytokines. Both pathomechanisms are known to contribute to graft rejection crises in vivo. Oxidative stress was induced in endothelial cell cultures with 10-200 microM buthionine sulfoximine. Western blotting showed a significant increase in the production of CMV-specific immediate early and late proteins in buthionine sulfoximine-treated cultures. Immunocytochemical staining suggested that this effect was caused by increased numbers of CMV antigen expressing cells (66% immediate early; 78%, late). Quantitative polymerase chain reaction for CMV-specific DNA and virus titration revealed that enhanced viral replication levels correlated with increased virion production. As a measure for the endothelial cell activation status, the surface expression of HLA-ABC and HLA-DR and adhesion molecules (ICAM-1, ELAM-1, VCAM-1) was quantified by fluorometric methods. Whereas oxidative stress alone did not modulate any surface molecule expression, the IFN-gamma-mediated expression of HLA-ABC and HLA-DR and the IL-1-mediated expression of ICAM-1, but not of ELAM-1 and VCAM-1 (IL-1 + TNF-alpha), was amplified. Interestingly, the amplification of HLA molecule expression was even higher in CMV-infected endothelial cells. This study provides evidence that oxidative stress contributes to the regulation of CMV replication, virus shedding, and the activation of endothelial cells by proinflammatory cytokines as it is observed in transplant recipients.

[Controlled reperfusion of the extremities for preventing local and systemic damage after prolonged ischemia. An experimental study with the swine model]. / Kontrollierte Extremitäten-Reperfusion zur Verminderung lokaler und systemischer Schäden nach mehrstündiger Ischämie. Eine experimentelle Studie am Schweinemodell.

Our previous studies in isolated rat hindlimbs using crystalloid perfusion solutions have shown that control of the initial reperfusion reduces postischemic complications. However, no experimental study has been undertaken to evaluate the concept of controlled limb reperfusion experimentally in an in-vivo blood-perfused model and to assess the local as well as systemic effects of normal blood reperfusion and controlled limb reperfusion. Of twenty pigs undergoing preparation of the infrarenal aorta and iliac arteries, six were observed for 7.5 hours and served as controls. Fourteen other pigs underwent 6 hours of complete infrarenal occlusion. Thereafter, embolectomy was stimulated in 8 pigs by removing the aortic clamp and establishing normal blood reperfusion at systemic pressure. In 6 other pigs, control of the composition of the reperfusate and control of the conditions of reperfusion was done during the first 30 min, followed by normal blood reperfusion. Six hours of infrarenal aortic occlusion lead to a severe decrease in high energy phosphates and muscle temperature and a slight increase in creating kinase (CK) and potassium in the systemic circulation. Normal blood reperfusion resulted in severe reperfusion injury: massive edema developed (80.6% vs. 76.6%, p < 0.0009), the tissue showed a marked decrease in oxygen consumption (7.3 +/- 1.1 vs. 14.3 +/- 2.5 mL )2/100 g/min, p < 0.02), glucose consumption (0.19 +/- 0.06 vs. 0.51 +/- 0.03 mg/100 g/min, p < 0.06), tissue ATP (18.3 +/- 1.9 vs. 36.1 +/- 0.9 mumol/g protein, p < 0.000001), total adenine nucleotides (26.3 +/- 2.6 vs. 45.8 +/- 1.5 mumol/g protein, p < 0.00001), muscle pH (5.9 +/- 0.1 vs. 7.3 +/- 0.1, p < 0.000006) and total calcium in the femoral vein (2. +/- 0.1 vs. 2.7 +/- 0.1 mmol/L, p < 0.002). Furthermore, a massive increase was seen in CK concentration (12,743 +/- 2,562 vs. 513 +/- 80 U/L, p < 0.0003), potassium (7.9 +/- 0.3 vs. 4.4 +/- 0.2 mmol/L, p < 0.000001) and muscle rigidity (60 +/- 11 vs. 122 +/- 1 degree, p < 0.00008). In sharp contrast, initial treatment of the ischemic skeletal muscle by controlled limb reperfusion resulted in normal water content (77.6 +/- 0.4 vs. 76.8 +/- 0.3%), oxygen consumption (13.2 +/- 1.6 vs. 14.9 +/- 3.2 mL O2/100 g/min), glucose consumption (0.58 +/- 0.18 vs. 0.46 +/- 0.11 mg/100 g/min), flow (5.4 +/- 1.1 vs. 4.6 +/- 4.6 +/- 0.5 mL/100 g/min) and muscle rigidity (106 +/- 4 vs. 122 +/- 1 degree). Furthermore, controlled limb reperfusion resulted in higher total adenine nucleotides content (78% vs. 57% of control), less tissue acidosis (6.6 +/- 0.2 vs. 5.9 +/- 0.1, p < 0.002), severely reduced CK release (2,618 +/- 702 vs. 12,743 +/- 2.562, p < 0.02) and potassium release (5.1 +/- 0.3 vs. 7.9 +/- 0.3 mmol/L, p < 0.0002) as compared to normal blood reperfusion. In conclusion this study shows that 6 hours of acute infrarenal aortic occlusion will result in a severe reperfusion injury (postischemic syndrome) if normal blood at systemic pressure is given in the initial reperfusion phase. In contrast, initial treatment of the ischemic skeletal muscle by controlled limb reperfusion reduces the metabolic, functional and biochemical alterations.

Reconstitution of bacteriorhodopsin and ATP synthase from Micrococcus luteus into liposomes of the purified main tetraether lipid from Thermoplasma acidophilum: proton conductance and light-driven ATP synthesis.

The archaebacterium Thermoplasma acidophilum is cultivated at 59 degrees C in a medium containing sulfuric acid of pH 2. The purified bipolar membrane spanning main phospholipid (MPL) of this organism can be used to produce stable liposomes of 100-500 nm in diameter either using a French pressure cell detergent dialysis or sonication. Despite a potassium diffusion potential of 186 mV very low ionic permeability of sonicated MPL liposomes was measured using the potassium binding fluorescent indicator benzofuran isophthalate PBF1, which measures net K+ uptake. The latter also remained very low, in the presence of the K(+) ionophore valinomycin and palmitic acid. Addition of valinomycin and the potent uncoupler carbonylcyanid-p-trifluormehoxyphenyl-hydrazone (FCCP), led to a stimulation in potassium uptake. The rate of proton flux can be calculated from the net K(+) uptake. Under these conditions MPL liposomes are 1-2 orders of magnitude less permeable than egg yolk lecithin vesicles. The difference in proton permeability becomes even more pronounced with increasing temperature, examined using the fluorescent pH indicator pyranine. Purified bacteriorhodopsin from Halobacterium halobium was reconstituted into MPL liposomes in order to study the light-driven proton uptake in 150 mM KCl following addition of valinomycin, gramicidin, FCCP and Triton X-100. The light-driven proton transport into the liposomes was increased 30-fold by addition of valinomycin decreased by gramicidin and FCCP, and abolished by Triton X-100. Co-reconstituted MPL proteoliposomes containing bacteriorhodopsin and ATP synthase from Micrococcus luteus were capable of light-driven ATP synthesis demonstrating the functional coupling of proton transport and nucleotide generation in liposomal MPL membranes.

Reperfusion injury in skeletal muscle: controlled limb reperfusion reduces local and systemic complications after prolonged ischaemia.

Previous studies in isolated limbs using crystalloid perfusion solutions have shown that control of the initial reperfusion reduces postischaemic complications. However, no experimental study has been undertaken to evaluate the concept of controlled limb reperfusion experimentally in an in vivo blood-perfused model and to assess the local as well as systemic effects of normal blood reperfusion and controlled limb reperfusion. Of 20 pigs undergoing preparation of the infrarenal aorta and iliac arteries, six were observed for 7.5 h and served as controls; 14 others underwent 6 h of complete infrarenal occlusion. Thereafter, embolectomy was simulated in eight pigs by removing the aortic clamp and establishing normal blood reperfusion at systemic pressure. In six other pigs, the composition of the reperfusate and the conditions of reperfusion were controlled during the first 30 min, followed by normal blood reperfusion. Some 6 h of infrarenal aortic occlusion leads to a severe decrease in high-energy phosphates and muscle temperature, together with a slight increase in creatine kinase and potassium in the systemic circulation. Normal blood reperfusion resulted in severe reperfusion injury: massive oedema developed, the tissue showed a marked decrease in oxygen consumption, glucose consumption, tissue ATP, total adenine nucleotides, muscle pH and total calcium in the femoral vein. Furthermore, a massive increase was seen in plasma creatine kinase concentration and potassium, together with the development of muscle rigidity. In sharp contrast, initial treatment of the ischaemic skeletal muscle by controlled limb reperfusion resulted in normal water content, oxygen consumption, glucose consumption, flow and muscle rigidity. Furthermore, controlled limb reperfusion resulted in higher total adenine nucleotides content, less tissue acidosis, markedly reduced creatine kinase release, and potassium release as compared with that of normal blood reperfusion. This study shows that 6 h of acute infrarenal aortic occlusion will result in severe reperfusion injury (postischaemic syndrome) if normal blood at systemic pressure is given in the initial reperfusion phase. In contrast, initial treatment of the ischaemic skeletal muscle by controlled limb reperfusion reduces the metabolic, functional and biochemical alterations.

Penetration of spin-labeled dihydrolipoate into the skin of hairless mice. Modification of epidermal and dermal polarity.

Electron paramagnetic resonance (EPR) imaging with the modulated field gradient technique is a novel method to investigate skin biophysical and biochemical properties employing specific nitroxide spin probes. Using this method, a distinct increase in polarity from epidermis towards lower dermis is observed with the spin label dit-butylnitroxide (DTBN). With proxylmaleimide a considerable increase in mobility is found, when epidermis is compared with dermal compartments. The effect of the natural antioxidant dihydrolipoate on skin membrane polarity was studied. Skin penetration of spin labeled dihydrolipoate was investigated by EPR imaging. The results indicate that dihydrolipoate also increases membrane polarity. The biophysical and biochemical changes in the epidermis and dermis as revealed by spatial imaging, provide indirect evidence for skin penetration of dihydrolipoate. This conclusion was supported by the finding that spin labeled derivatives of dihydrolipoate and lipoate were detected inside epidermis and dermis by EPR imaging. This study demonstrates the feasibility of EPR imaging to investigate pharmacodynamic and pharmacokinetic properties of spin labeled drugs in skin.

Chemiluminescence in semen of infertile men.

Luminol-dependent chemiluminescence (CL) can be used to determine the production of reactive oxygen species (ROS) by cells. Enhanced formation of ROS in human semen was reported to be of pathological significance for a disturbed sperm function. To investigate incidence of elevated CL-signals in semen samples and their correlation to conventional semen parameters, CL-signals in the semen of both 49 consecutive infertile men and 20 controls were measured. Semen was analysed according to WHO-criteria including bovine mucus-penetration- and water-test. A CL-signal of 1.5 x 10(5) counts min-1/2 x 10(6) spermatozoa was considered to be the upper normal limit. The CL in infertile men's semen was elevated with statistically significant differences in oligozoospermia patients/controls (P < 0.0001) and normozoospermia patients/controls (P < 0.05). In the group with elevated CL-signals, a higher percentage of spermatozoa with a pathologic morphology was detected (P = 0.05). In the groups with pathologic results of eosin- and water-tests, the CL-counts were elevated (P < 0.006; P < 0.03). The spermatozoa motility in the group with elevated CL-counts was significantly reduced after 4 h (P < 0.05). The CL-signals correlated inversely with the results of the bovine mucus-penetration-test (r = -0.67), P < 0.0001). In conclusion, semen samples of 28% of our patients showed elevated CL-signals; these were associated with pathological results of membrane integrity-tests. The negative correlation of CL with the results of Penetrak-test reflects its importance to depict the functional capacity of spermatozoa.