On the use of the spin labeling technique in the study of erythrocyte membranes

ESR spectra and scanning electron micrographs of human erythrocytes spin labeled with the conventional stearic acid nitroxide substituted at the 5-position have been obtained over a range of label-to-lipid ratios. While morphological changes as previously reported (Bieri V.G.; Wallach D.F.H.; Lin P.S. (1974) Proc. Natl. Acad. Sci. U. S. 71, 4797–4801) are reproduced, it is shown that at label-to-lipid ratios of 1 : 10 or less the basic ESR spectrum is not significantly affected. At low label concentrations the spin labeling technique is a viable one and can be used to investigate membrane properties.     

On the use of the spin labeling technique in the study of erythrocyte membranes.

ESR spectra and scanning electron micrographs of human erythrocytes spin labeled with the conventional stearic acid nitroxide substituted at the 5-position have been obtained over a range of label-to-lipid ratios. While morphological changes as previously reported (Bieri, V. G., Wallach, D. F. H. and Lin, P. S. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4797-4801) are reproduced, it is shown that at label-to-lipid ratios of 1:10 or less the basic ESR spectrum is not significantly affected. At low label concentrations the spin labeling technique is a viable one and can be used to investigate membrane properties.     

Comparison of different hepatocyte cell lines for use in a hybrid artificial liver model

Selection of a cell line suitable for a hybrid artificial liver model employing cellulose porous beads (CPBs) was investigated. Hep G2 cells grown in a culture dish exhibited appreciably higher ureogenesis and gluconeogenesis activities than those grown in CPBs. SEM observation of CPBs revealed marked difference in the distribution of attached cells from one bead to another, and showed that almost all the cell-bearing micropores were completely packed with cells. With the aim of selecting a cell line not prone to excessive aggregation and which grows moderately so as not to fill up the micropores, cells of 6 cell lines, HLE, HLF, Hep 3B, PLC/PRF/5, Huh 7 and Hep G2, were cultivated in dishes. Hep G2, HLE, and HLF increased to 5 × 10⁵ cells/cm², whereas PLC/PRF/5 grew only to 5 × 10⁴, and Hep 3B and Huh 7 up to 2 × 10⁴ cells/cm². The specific activities of ureogenesis and gluconeogenesis of Huh 7 were the highest among the lines tested - 42- and 7-fold those of Hep G2, respectively. When the 6 cell lines were grown in a submerged culture with 0.6 g/l of CPBs, Huh 7 had the lowest cell concentration of 0.54 × 10⁶ cells/ml, and the highest activities of ammonia consumption and urea and glucose production (1.38 μ mol NH₃, 99 nmol urea, and 14.5 nmol glucose/10⁶cells/h). Consequently, Huh 7 is considered to be a suitable cell line for use in the development of an artificial liver model employing porous beads. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparison of different algal carbon estimates by use of the Droop-model for nutrient limited growth

Three different estimates of algal carbon were derived fromfield experiments in plastic enclosures and tested as measureof algal biomass in the growth model of Droop. Fresh weightderived carbon and total paniculate organic carbon correctedfor detritus were both found to behave as good estimates ofalgal biomass in the model, while total paniculate organic carbondid not. The study also provided results which suggested thatthe relation between growth rate and endogenous phosphorus differedfor two species involved. Growth was maintained at lower Q_p-ratiosin Staurastrum dominated communities compared with communitiesconsisting mainly of Anabaena flos-aquae.     

The use of cis-parinaric acid to determine lipid peroxidation in human erythrocyte membranes. Comparison of normal and sickle erythrocyte membranes

The recently developed parinaric acid assay is shown to offer possibilities for studying peroxidation processes in biological membrane systems. Taking the human erythrocyte membrane as a model, several initiating systems were investigated, as well as the effect of residual hemoglobin in ghost membrane preparations. The effectivity of a radical generating system appeared to be strongly dependent upon whether radicals are generated at the membrane level or in the water phase. Thus, cumene hydroperoxide at concentrations of 1.0-1.5 mM was found to be a very efficient initiator of peroxidation in combination with submicromolar levels of hemin-Fe3+ as membrane-bound cofactor. In combination with cumene hydroperoxide, membrane-bound hemoglobin appeared to be about 6-times more effective in promoting peroxidation than hemoglobin in the water phase. Results comparing the behaviour of normal and sickle erythrocyte ghost suspensions in the peroxidation assay suggest that the increased oxidative stress on sickle erythrocyte membranes could be due to enhanced membrane binding of sickle hemoglobin, but also partly to a characteristically higher capability of sickle hemoglobin to promote peroxidation. The order of peroxidation-promoting capabilities that could be derived from the experiments was hemin greater than sickle hemoglobin greater than normal hemoglobin.     

Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities

ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the facade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng microl(-1), while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.     

A proposed protocol for the standardized preparation of PRF membranes for clinical use

Upon clinical application, thick platelet-rich fibrin (PRF) is usually compressed to fit the implantation site. However, it is speculated that the preservation of platelets and plasma content depends on the compression methods used. To accurately evaluate the clinical outcome of PRF, the preparation protocol should be standardized. Freshly prepared PRF clots were compressed into a thin membrane by our novel PRF compression device. The localization of platelets was examined by SEM and immunostaining. Growth factor levels were evaluated by bioassays and cytokine-antibody array techniques. The angiogenic activity was examined by the chick chorioallantoic membrane assay and the scratch assay using HUVEC cultures. Platelets were concentrated on the surface of the region adjacent to the red thrombus and this region was subjected to the experiments. Compared to the PRF membrane compressed by dry gauze (G-PRF), the preservation of the plasma content, 3D-fibrin meshwork, and platelets was more intact in the compressor-prepared PRF membrane (C-PRF). Among the growth factors tested, C-PRF contained PDGF isoforms at higher levels, and significantly stimulated cell proliferation and neovascularization. C-PRF may be useful for grafting while minimizing the loss of bioactive factors. This C-PRF preparation protocol is proposed as a standardized protocol for PRF membrane preparation.     

Comparison of two protocols for the treatment of retained fetal membranes in dairy cattle

Two protocols for the treatment of retained fetal membranes in dairy cattle were evaluated in a field trial. Cows that retained the fetal membranes for more than 12h were assigned to two treatment groups in an alternating order. In both groups rectal temperature was measured daily for 10 days after enrollment. In Group 1 (n=35) cows with a rectal temperature >or=39.5 degrees C received a systemic antibiotic treatment with 600mg ceftiofur intramuscularly on three consecutive days. No manual removal of the fetal membranes or intrauterine treatment was conducted. In case of elevated temperature of >or=39.5 degrees C on Day 3 treatment was conducted for another 2 days. In Group 2 (n=35) cows received a local antibiotic treatment (2500 mg ampicillin, 2500 mg cloxacillin) and an attempt was made to remove the fetal membranes manually. In case of a rectal temperature >or=39.5 degrees C 6000 mg of ampicillin were administered intramuscularly. Treatment was repeated on three consecutive days. If temperature did not decrease below 39.5 degrees C systemic treatment was extended for another 2 days.During 10 days of observation 33 and 34 cows showed fever, i.e. a body temperature >or=39.5 degrees C in Groups 1 and 2, respectively (94.3 versus 97.1%). The proportion of cows considered as cured (temperature <39.5 degrees C on Day 10 after enrollment) was 65.7 and 68.6% in Groups 1 and 2, respectively. All cows showed signs of chronic inflammation of the genital tract on Day 14 after calving. Within 4 weeks postpartum three (8.6%) and four (11.4%) cows were culled in Groups 1 and 2, respectively. Days to first service and days open did not differ significantly between the groups. Proportion of cows pregnant on Day 200 postpartum was 71.4 and 54.3% for Groups 1 and 2, respectively (P>0.05). Results indicate that treatment of retained fetal membranes without intrauterine manipulation and treatment can be as effective as conventional treatment including detachment and local antibiotic treatment.     

Adaptation of the inert gas FRC technique for use in heavy exercise

We automated the inert gas rebreathe technique for measurement of end-expiratory lung volume (EELV) during heavy exercise. We also assessed the use of two gas tracers (He and N2) vs. a single gas tracer (He) for measurement of this lung volume and compared the two-tracer EELV to changes in the inspiratory capacity (defined with transpulmonary pressure) and shifts in the end-expiratory pressure from rest through heavy exercise. A computer program switched a pneumatic valve when flow crossed zero at end expiration and defined points in the He and N2 traces for calculation of EELV. An inherent delay of the rebreathing valve (50 ms) caused virtually no error at rest and during light exercise and an error of 74 +/- 9 ml in the EELV at peak inspiratory flow rates of 4 l/s. The measurement of EELV by the two gas tracers was closely correlated to the single-gas tracer measurement (r = 0.97) but was consistently higher (120 +/- 10 ml) than when He was used alone. This difference was accentuated with increased work rates (2-5% error in the EELV, rest to heavy exercise) and as rebreathe time increased (2-7% error in the EELV with rebreathe times of 5-20 s for all work loads combined). The double-gas tracer measurement of EELV agreed quite well with the thoracic gas volume at rest (P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of methods for assessing integrity of equine sperm membranes

Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = −3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = −11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.     

Comparison of methods for extracting thylakoid membranes of Arabidopsis plants

Robust and reproducible methods for extracting thylakoid membranes are required for the analysis of photosynthetic processes in higher plants such as Arabidopsis. Here, we compare three methods for thylakoid extraction using two different buffers. Method I involves homogenizing the plant material with a metal/glass blender; method II involves manually grinding the plant material in ice‐cold grinding buffer with a mortar and method III entails snap‐freezing followed by manual grinding with a mortar, after which the frozen powder is thawed in isolation buffer. Thylakoid membrane samples extracted using each method were analyzed with respect to protein and chlorophyll content, yields relative to starting material, oxygen‐evolving activity, protein complex content and phosphorylation. We also examined how the use of fresh and frozen thylakoid material affected the extracts' contents of protein complexes. The use of different extraction buffers did not significantly alter the protein content of the extracts in any case. Method I yielded thylakoid membranes with the highest purity and oxygen‐evolving activity. Method III used low amounts of starting material and was capable of capturing rapid phosphorylation changes in the sample at the cost of higher levels of contamination. Method II yielded thylakoid membrane extracts with properties intermediate between those obtained with the other two methods. Finally, frozen and freshly isolated thylakoid membranes performed identically in blue native‐polyacrylamide gel electrophoresis experiments conducted in order to separate multimeric protein supracomplexes.