Complement Factor H-Related Protein 3 Serum Levels Are Low Compared to Factor H and Mainly Determined by Gene Copy Number Variation in CFHR3

The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein.     

β-adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

Specific binding of [¹²⁵I]-(−)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (K_d=5.3±0.9 pmol/l and R_T=78±7fmol/g tissue). The β₁-selective antagonists atenolol and LK 203-030 inhibited specific [¹²⁵I]-(−)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous β₂-adrenoceptor population. In one subject using LK 203-030 a small β-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pK_H- and pK_L- values for the high and low affinity sites were 8.0±0.2 and 5.9±0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD₂-value of 6.63±0.19.     

Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO₃ was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed.     

An inflatable minirhizotron system for root observations with improved soil/tube contact

Commonly used minirhizotrons consisting of a transparent tube inserted into the soil seldom attain good contact between the tube and the soil, which leads to root growth occurring in a gap rather than in the soil. A new system is described involving an inflatable flexible rubber wall, made from a modified motorcycle tube. Pressure ensures a proper tube/soil contact so that the environmental circumstances for root growth along the tube more closely correspond to those in the undisturbed soil. Before the endoscope slide is introduced into the minirhizotron for taking pictures, the inflatable tube is removed, so that there is no-often opaque-wall between the endoscope and the roots. This improves the picture quality and facilitates the analysis of root images.     

Hydroxamate-Stimulated O(2) Uptake in Roots of Pisum sativum and Zea mays, Mediated by a Peroxidase : Its Consequences for Respiration Measurements

Low concentrations of salicylhydroxamic acid (<5 millimolar) stimulate O₂ uptake in intact roots of Pisum sativum. We demonstrate that the hydroxamate-stimulated O₂ uptake does not reside in the mitochondria. We also show that the hydroxamate-stimulated O₂ uptake is due to the activation of a peroxidase catalyzing reduction of O₂. This peroxidase, which can use both NADH and NADPH as a substrate, is stimulated by low concentrations of monophenols, e.g. salicylhydroxamic acid and 2-methoxyphenol. It is inhibited by high (20 millimolar) concentrations of salicylhydroxamic acid, cyanide, and scavengers of the superoxide free radical ion, e.g. ascorbate, gentisic acid, and catechol. In the presence of gentisic acid, O₂ uptake by intact pea roots was no longer stimulated by low concentrations of salicylhydroxamic acid. The consequence of the present finding for in vivo respiration measurements is that the use of low concentrations of salicylhydroxamic acid and uncoupler is reliable only in the presence of a suitable superoxide free radical scavenger which prevents activation of the peroxidase. It also confirms that high concentrations of salicylhydroxamic acid (20-25 millimolar) can be safely used in short-term experiments to assess the activity of the alternative path in intact roots.     

Density fluctuations in populations (1982–1986) and biological observations ofPotamopyrgus Jenkinsi in two trophically differing lakes

The hydrobiid snailPotamopyrgus jenkinsi (E.A. Smith), characterized by parthenogenesis and ovovivipary, was quantitatively sampled monthly between June, 1982, and December, 1986, on sandy bottoms in the shallow zones of the meso-oligotrophic Lake Maarsseveen I and the eutrophic Lake Maarsseveen II. The snail demonstrated a very clumped distribution in both lakes. The mean numbers of juveniles and adults taken together fluctuated strongly. Organisms in Lake I showed relatively high densities (up to 25,000 per m²) in 1982, followed by a sudden drop to values approaching zero in December, 1982, with a subsequent rapid increase in densities, fluctuating between 2,000 and 200 per m². In Lake II, densities of snails fluctuated between 13,000 and 300 per m² with decreases in the spring of 1985 and 1986. The various types of decreases in the lakes are extensively discussed, but no explanation is presently available. The reduction in Lake I was of catastrophic proportions, but the speed of recovery of the population was remarkable.Floating was observed only in Lake I, and only during the occurrence of the highest densities on the sediment. Burrowing behaviour was very common, but strongly suppressed under an uninterrupted dark regime. A shift of temperature from 15 to 22°C had the same effect. A number of submerged macrophyte species from Lake I proved to attractP. jenkinsi in the absence of sandy substrate, though these plants were only covered by the snail during the period of the highest densities in 1982. Temperatures of 20°C or lower were well tolerated, unlike temperatures of 25 and 30°C. Growth was distinct at 10, 15 and 20°C. Keeled individuals were encountered in much higher numbers in Lake I than in Lake II.     

13C-NMR studies on the influence of pH and nitrogen source on polyol pool formation in Aspergillus nidulans

Abstract The composition of the polyol pools in Aspergillus nidulans mycelium during active growth on sucrose depends strongly on pH. At pH 2.5, only mannitol is present. A comparison between nitrate- and ammonium-grown cultures shows stimulation of the arabitol content with nitrate a former nitrogen source. When starved mycelium is incubated either with natural-abundance or ¹³C-enriched glucose, label appears rapidly in mannitol and arabitol, regardless of the nitrogen source or the pH used.     

Effect of pKM101 on cell killing and specificity of mutation induction by cis-diaminedichloroplatinum(II) in Escherichia coli K-12.

Cell killing and mutation induction in the lacI gene of Escherichia coli by cis-Pt(NH3)2Cl2 were studied in cells with different repair capacities, with and without pKM101. The presence of the plasmid pKM101 made repair-proficient cells more susceptible to killing by cis-Pt(NH3)2Cl2 and strongly enhanced mutation induction by that compound. Both effects were shown to be dependent upon excision repair. Characterization of the induced mutations in the lacI gene after cis-Pt(NH3)2Cl2 treatment of E. coli cells, by the LacI system, revealed that the mutagenic specificity of the Pt compound was strongly influenced by the presence of the pKM101 plasmid. With pKM101, 23% of the induced amber and ochre mutations resulted from substitutions at AT base pairs, whereas these mutations were hardly induced in cells without pKM101. These results suggest that pKM101-induced repair differs from normal SOS repair.