On-line measurements of culture fluorescence: Method and application

Summary In this paper a new probe allowing the measurement of NAD(P)H-dependent culture fluorescence in a bioreactor is presented. This sterilizable probe can be inserted in every bioreactor using a standard fitting of 25 mm. Under well defined conditions high specificity and sensitivity as well as high stability are further advantages of this probe. Application examples are given to demonstrate the operation possibilities of this fluorescence probe. In batch growth the culture fluorescence can be used for on-line estimation of biomass concentration. Metabolic alterations due to substrate of oxygen deficiency can easily be detected by fluorometric measurements. In kinetic studies the fluorescence probe is of great use because of a very small time delay.     

Radioimmunoassay of 2-hydroxyestrone

Under the protection of ascorbic acid a 2-hydroxyestrone bovine serum albumin conjugate was prepared containing intact 2-hydroxyestrone as determined by gas chromatographymass spectrometry. Using this antigen highly specific antibodies were raised in rabbits. Cross-reactivity for 2-hydroxyestradiol and 2-hydroxyestriol was 26 and 4.5%, respectively. An assay procedure of 2-hydroxyestrone in human plasma is described. Using special precautions the assay allows the determination of 2-hydroxyestrone in plasma samples of women (50–95 pg/ml), pregnant women (105–220 pg/ml), men (45–65 pg/ml) and children (20–40 pg/ml).     

Characteristics of the F52 protein, a MARCKS homologue.

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.     

Genetic control of human apolipoprotein E polymorphism: Comparison of one-and two-dimensional techniques of isoprotein analysis

Summary Genetic polymorphism of human apolipoprotein E (apo E) has previously been demonstrated by one-dimensional isoelectric focusing (Utermann et al. 1977b) and by two-dimensional electrophoresis of apolipoproteins (Zannis et al. 1981), but the relationship between the results obtained by these methods remained unclear. We therefore performed comparative phenotyping by one-dimensional and two-dimensional electrophoresis. Apoproteins from very low-density lipoproteins (apo VLDL) prepared by ultracentrifugation or from an apo Erich lipoprotein fraction prepared by heparin/Mg⁺⁺ precipitation, were used as a source of apo E. Six common phenotypes designated apo E-4/4, apo E-N/N, apo E-D/D, apo E-4/N, apo E-4/D, and apo E-N/D were differentiated irrespective of the technique used or the source of apolipoproteins, but the two-dimensional electrophoresis of apo VLDL and apo VLDL which had been treated with neuraminidase was the key for the correct genetic interpretation of those phenotypes exhibiting the E4 isoform of the protein. Each phenotype is characterized by the presence of either one or two of three major isoforms E2, E3, and E4 and by the presence of several minor sialylated forms of these proteins (apo E_s) that have higher apparent molecular weights. The unsialylated major isoform apo E2 does not only differ in charge but also has a higher apparent mol.wt. (about 34,500) than the major isoforms apo E3 and apo E4 (mol. wt. about 33,000). Family studies including 90 matings with a total of 203 offspring confirmed the genetic one locus model of Zannis et al. (1981). Apo E phenotypes are controlled by three autosomal codominant alleles apo E^d, apo Eⁿ, and apo E⁴ that specify for the E2, E3, and E4 isoforms respectively. Phenotypes apo E-D/D,-N/N, and-4/4 represent homozygotes and phenotypes apo E-4/N,-4/D, and-N/D heterozygotes for these alleles.The frequencies of apo E alleles in 1031 blood donors were apo E⁴=0.150, apo Eⁿ=0.773, and apo E^d=0.077. Homozygosity for the allele apo E^d is associated with hyperlipoproteinemia type III. Hence a large number of the population (about 1%) are at risk for this specific lipoprotein disorder that is associated with premature atherosclerosis and xanthomatosis.     

Territory Defence in Black Redstarts,Phoenicurus ochruros: Effects of Intruder and Owner Age?

European black redstarts, Phoenicurus ochruros, vigorously defend all-purpose territories and exhibit delayed plumage maturation, most subadult males looking exactly female-like in their first breeding season. We tested the hypotheses that such dull subadult male plumages are beneficial in order to reduce aggression of adult males either by deception or by honest signalling of subordinance status, and that, in turn, conspicuous (adult) plumage colorations are able to intimidate contenders because they act as a signal of fighting ability and aggressive motivation. Adult and dull yearling black redstart territory owners were confronted with intruders mimicked by stuffed mounts of either a conspicuous adult or a dull subadult male. Our results do not support the hypotheses tested: dull plumages of young intruders did not reduce aggression from adult territory owners and aggressiveness towards intruders was significantly higher in yearling territory owners as compared with adult owners. Conspicuous intruders did not deter dull territory owners and we found no indications that conspicuous male coloration is a signal of fighting ability or aggressive motivation in this species. Instead, the amount of aggressive response to intruders showed considerable individual variance within age classes regardless of the plumage of the intruder and the status of the owner.     

Peptide conformations--49(1): synthesis and structure-activity relationships of side chain modified peptides of cyclo(-D-Pro-Phe-Thr-Lys-Trp-Phe.)

Cyclic hexapeptide analogues representing the modified retro sequence of the amino acid residues 7-11 of natural somatostatin are known to protect liver cells from phalloidin poisoning. To determine the influence of steric, lipophilic, and charge effects on (a) the conformation of the backbone and the aromatic side chains and (b) the biological response, the side chains of Phe2, Lys4, and Phe6 of cyclo(-D-Pro1-Phe2-Thr3-Lys(Z)4-Trp5-Phe6-), 1a, one of the most active peptides found so far, were modified by various residues. The discussion of conformationally relevant parameters proves that neither backbone conformations nor populations of aromatic side chain rotamers were altered by these substitutions. The potency of these derivatives in a cytoprotection assay varies by at most one order of magnitude (more or less active than the parent peptide 1a). A qualitative evaluation of lipophilic, steric, and charge effects reveals the dominance of lipophilic effects of aromatic residues; the most potent compounds contain aromatic substructures in the side chain of Lys4.