Demonstration of chromosome replication by BrdU antibody technique and electron microscopy

Summary We describe the use of different reporter groups in visualizing replication patterns on metaphase chromosomes after BrdU incorporation by the BrdU antibody technique for the electron microscope. There is an inverse correlation between the density of the label and the size of the reporter particles. This observation alludes to stereo problems interfering with the access of the labeled antibodies into the chromatin. The use of silver enhancement enables easy detection of 1-nm and 5-nm gold particles, which make replication patterns visible in the electron microscope as does the diaminobenzidine/ H₂O₂ reaction. Possible consequences for the demonstration of replication patterns and for nonradioactive DNA in situ hybridization are discussed.     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a pan-epithelial monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation     

Genetics of the quantitative Lp(a) lipoprotein trait

The Lp(a) lipoprotein is a complex particle composed of a low density lipoprotein (LDL)-like lipoprotein and the disulfide bonded Lp(a) glycoprotein. The complex represents a quantitative genetic trait. SDS gel electrophoresis under reducing conditions of sera followed by immunoblotting with affinity-purified polyclonal anti-Lp(a) demonstrated inter- and intra-individual size heterogeneity of the glycoprotein with apparent Mr in the range 400-700kDa. According to their relative mobilities compared to apo B-100 the Lp(a) patterns were categorized into phenotypes F, B, S1, S2, S3 und S4 and into the respective double-band phenotypes. This size heterogeneity seems to be controlled by multiple alleles designated LpF, LpB, LpS1, LpS2, LpS3, LpS4 and a null allele (LpO) at a single locus. Phenotype frequencies observed in 441 unrelated subjects were in good agreement with those expected from the genetic hypothesis. Comparison of Lp(a) lipoprotein concentrations in the different phenotypes revealed a highly significant association of phenotypes B, S1 and S2 with high, and phenotypes S3 und S4 with intermediate Lp(a) concentrations. A third mode is represented by the null phenotype were no Lp(a) band is detected upon immunoblotting and Lp(a) lipoprotein is low or absent. We conclude that the same gene locus is involved in determining Lp(a) glycoprotein phenotype and Lp(a) lipoprotein concentrations in plasma. This major gene seems to be the Lp(a) glycoprotein structural gene locus.     

Genetics of the quantitative Lp(a) lipoprotein trait

Summary Lp(a) glycoprotein exhibits an apparent size polymorphism that is associated with genetically controlled Lp(a) lipoprotein concentrations in plasma (Utermann et al. 1988). We have tested the hypothesis that this polymorphism is genetically controlled by studying 15 matings with a total of 44 offspring. This confirmed our conclusion that Lp(a) types are controlled by a series of codominant alleles Lp^F, Lp^B, Lp^S1, Lp^S2, Lp^S3 and Lp^S4 and by a null allele Lp^o. Together with the data from the accompanying paper this indicates that the structural gene for the Lp(a) protein is the major gene locus determining Lp(a) lipoprotein concentrations in plasma.     

Simultaneous saccharification and protein enrichment fermentation of sugar beet pulp

Summary A product with 40 % protein content was obtained from sugar beet pulp (1.25–2.0 mm) in 48 h one stage (simultaneous) saccharification/fermentation process under optimized conditions using a specific enzyme mixture andCandida tropicalis strain, also saving about 40 % enzymes in comparison to a 2-stage process.     

Selection of preculture conditions for solid state fermentation of sugar beet pulp

Summary For the protein upgrading of sugar-beet pulp in solid state fermentation byTrichoderma reesei andFusarium oxysporum, serveral conditions were studied to prepare an economical preculture for large scale process. The best performance was shown by a preculture obtained in 24 h from 1.5 % molasses solution at pH 4.5–5.0 with 1.0 % milled beet pulp.     

Production of tumor necrosis factor by rIFN-gamma-primed C3H/HeJ (Lpsd) macrophages requires the presence of lipid A-associated proteins

Previous studies have shown that the activation of murine macrophages to a fully tumoricidal state requires that specific environmental signals be delivered to the macrophage in a step-wise manner: a "priming" signal first renders the macrophage stimulated, but not cytolytic. The addition of a second or "trigger" signal to the primed macrophage results in tumoricidal activity. One potent priming signal has been identified as IFN-gamma and one often used trigger signal for endotoxin-responsive (Lpsn) macrophages is LPS. In contrast to LPS-responsive macrophage, rIFN-gamma-primed C3H/HeJ (Lpsd) macrophages fail to become cytolytic in response to protein-free, phenol-water-extracted LPS preparations, but become tumoricidal when exposed in vitro to protein-rich butanol-extracted LPS or purified lipid A-associated proteins. Further characterization of the activation requirements of the C3H/HeJ macrophages revealed that for optimal elaboration of TNF in vitro, two signals were also required: rIFN-gamma and a second signal that contained LAP. C3H/HeJ macrophages macrophages primed with rIFN-gamma failed to produce TNF in response to any concentration of protein-free phenol-water extracted LPS, even when supernatants were concentrated before assaying for functional activity in a standard TNF L929 fibroblast assay. Although exposure of rIFN-gamma-primed C3H/HeJ macrophages to LAP resulted in a fully tumoricidal state equivalent to that exhibited by C3H/OuJ macrophages, the levels of TNF produced remained discrepant. Under identical conditions, C3H/OuJ macrophages produced approximately fivefold more TNF (11,776 U/ml) than C3H/HeJ macrophages (2,399 U/ml). This suggests that although C3H/HeJ macrophages can respond functionally in a "normal" manner given the correct signals, they remain quantitatively deficient in the production of certain proteins. In this system, the elaboration of TNF and macrophage-mediated tumor cell lysis were shown to be dissociable events. The tumor target used in these studies (P815) was shown to be resistant to as much as 40,000 U/ml of purified rTNF. In addition, C3H/OuJ macrophage cultures exposed to LPS only (which resulted in the production of high levels of TNF), failed to lyse these targets. Lastly, anti-mouse TNF antibody added to macrophage cultures had no effect on the induction of tumor cell lysis.     

Permeabilization of cultivated plant cells by electroporation for release of intracellularly stored secondary products

Summary Plant cell suspension cultures producing secondary metabolites have been permeabilized for product release by electroporation. The two cell cultures studied, i.e. Thalictrum rugosum and Chenopodium rubrum, require about 5 and 10 kV cm^–1, respectively, for complete permeabilization (release of all the intracellularly stored product). The number of electrical pulses and capacitance used had a relatively limited effect on product release while the viability of the cells was strongly influenced by the latter. Conditions for complete product release resulted in total loss of viability of the cells after treatment. The release of product from immobilized cells was also achieved by electroporation. Cells entrapped in alginate required less voltage for permeabilization than free or agarose entrapped cells.     

Die Ölblumensymbiosen – Parallelismus and andere Aspekte ihrer Entwicklung in Raum and Zeit1, 2

The oil-bee/oil-flower relationships: parallelism and other aspects of their evolution in space and time A survey is given of our present knowledge and existing hypotheses concerning the biogeography, history, and phylogeny of plant taxa yielding fatty oil as a floral reward, and of the bee genera involved in their pollination. Four syngenetic complexes of the symbiosis arose convergently: The neotropical, the paleotropical, the holarctic, and the capensic complex. On the basis of the mutual structural adaptations of bees and flowers it is concluded that, in addition, parallelism within related groups as a result of a common tendency to develop the respective organs, has played an important role in the evolution of the oil-based floral interrelationships.