Opalinidae in African Anura. III. Genus Cepedea

During 1988 and 1989, 409 specimens of southern African Anura comprising 50 species in 9 families were checked for opalinids in the cloaca. Cepedea acuta n. sp. was found in six of 19 Tomopterna cryptotis and four of seven T. krugerensis (Ranidae); C. affinis (Nazaretskaja, 1992) in two of four Afrixalus aureus, two of four Hyperolius horstocki, 23 of 42 H. marmoratus, one of seven H. pusillus, two of six H. semidiscus, and eight of 12 H. tuberilinguis (Hyperoliidae); C. magna Metcalf, 1923 in one of 20 Bufo garmani, eight of 33 B. gutturalis and two of nine B. rangeri (all Bufonidae), one of two Heleophryne natalensis (juveniles) (Heleophrynidae), four of six Kassina maculata, three of seven K. senegalensis and two of six Semnodactylus wealii (all Hyperoliidae), three of five Phrynomerus bifasciatus (Microhylidae), three of 12 Phrynobatrachus natalensis, 11 of 19 Tomopterna cryptotis, 13 of 14 T. delalandii, one of seven T. krugerensis and one of six T. natalensis (all Ranidae), and four of five Chiromantis xerampelina (Rhacophoridae); Cepedea vanniekerkae n. sp. in one of 19 Tomopterna cryptotis. It is suggested that the Ranidae (in particular the genus Tomopterna) and the Hyperoliidae (in particular the genus Hyperolius) are among the major carriers of Cepedea in the Afrotropical Region.     

Site-directed mutagenesis of the yeast PRP20/1SRM1 gene reveals distinct activity domains in the protein product

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.     

DNA fingerprinting of red clover (Trifolium pratense L.) with Jeffrey's probes: detection of somaclonal variation and other applications

Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6 detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F₁ plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within (0.417≤V≤0.548) cultivars.     

Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development

A gene exhibiting homology to the polygalacturonases of several species, including tomato and Oenothera, has been shown by RNA dot-blot analysis and in situ hybridization experiments to be expressed post-first microspore mitosis in maize. A 2.87 kbp section of the promoter fused to E. coli β-glucuronidase (uidA) coding sequence conferred the correct spatial and temporal expression in transgenic tobacco plants. However, low levels of expression were detected in other tissues, and in particular in the tissues surrounding the vascular branch points of leaf nodes. The maize polygalacturonase gene is one member of a highly conserved gene family. The lack of detectable expression in sporophytic tissues and the isolation of a number of related cDNAs from maize suggests that all expressed members of this family show the same spatial and temporal regulation.     

The agony of choice: Impact of the host animal species on the enzyme-linked immunosorbent assay performance for host cell protein quantification

Host cell proteins (HCPs) are inevitable process-related impurities in biotherapeutics commonly monitored by enzyme-linked immunosorbent assays (ELISAs). Of particular importance for their reliable detection are the anti-HCP polyclonal antibodies (pAbs), supposed to detect a broad range of HCPs. The present study focuses on the identification of suitable host animal species for the development of high-performance CHO-HCP ELISAs, assuming the generation of pAbs with adequate coverage and specificity. Hence, antibodies derived from immunization of sheep, goats, donkeys, rabbits, and chickens were compared concerning their amount of HCP-specific antibodies, coverage, and performance in a sandwich ELISA. Immunization of sheep, goats, donkeys, and rabbits met all test criteria, whereas the antibodies from chickens cannot be recommended based on the results of this study. Additionally, a mixture of antibodies from the five host species was prepared to assess if coverage and ELISA performance can be improved by a multispecies approach. Comparable results were obtained for the single- and multispecies ELISAs in different in-process samples, indicating no substantial improvement for the latter in ELISA performance while raising ethical and financial concerns.     

Phylogeny of Saxifraga section Saxifraga subsection Arachnoideae (Saxifragaceae) and the origin of low elevation shade-dwelling species

Saxifraga section Saxifraga subsection Arachnoideae is a lineage of 12 species distributed mainly in the European Alps. It is unusual in terms of ecological diversification by containing both high elevation species from exposed alpine habitats and low elevation species from shady habitats such as overhanging rocks and cave entrances. Our aims are to explore which of these habitat types is ancestral, and to identify the possible drivers of this remarkable ecological diversification. Using a Hybseq DNA-sequencing approach and a complete species sample we reconstructed and dated the phylogeny of subsection Arachnoideae. Using Landolt indicator values, this phylogenetic tree was used for the reconstruction of the evolution of temperature, light and soil pH requirements in this lineage. Diversification of subsection Arachnoideae started in the late Pliocene and continued through the Pleistocene. Both diversification among and within clades was largely allopatric, and species from shady habitats with low light requirements are distributed in well-known refugia. We hypothesize that low light requirements evolved when species persisting in cold-stage refugia were forced into marginal habitats by more competitive warm-stage vegetation. While we do not claim that such competition resulted in speciation, it very likely resulted in adaptive evolution.     

Comparative LCA studies of simulated HMF biorefineries from maize and miscanthus as an example of first- and second-generation biomass as a tool for process development

5-Hydroxymethylfurfural (HMF) is a versatile platform chemical for a fossil free, bio-based chemical industry. HMF can be produced by using fructose as a feedstock. Using edible, first-generation biomass to produce chemicals has been questioned in terms of potential competition with food supply. Second-generation biomass like miscanthus could be an alternative. However, there is a lack of information if second-generation lignocellulosic biomass is a more sustainable feedstock to produce HMF. Therefore, a life cycle assessment was performed in this study to determine the environmental impacts of HMF production from miscanthus and to compare it with HMF from high-fructose corn syrup (HFCS). HFCS from either Hungary or Baden-Württemberg (Germany) was considered. Compared to the HFCS biorefineries the miscanthus concept is producing less emissions in all impact categories studied, except land occupation. Overall, the production and usage of second-generation biomass could be especially beneficial in areas where the use of N fertilizers is restricted. Besides, conclusions for the further development of the on-farm biorefinery concept were elaborated. For this purpose, process simulations from a previous study were used. Results of the previous study in terms of TEA and the current LCA study in terms of environmental sustainability indicate that the lignin depolymerization unit in the miscanthus biorefinery has to be improved. The scenario without lignin depolymerization performs better in all impact categories. The authors recommend to not further convert the lignin to products like phenol and other aromatic compounds. The results of the contribution analyses show that the major impact in the HMF production is caused by the auxiliary materials in the separation units and the required heat. Further technical development should focus on efficient heat as well as solvent use and solvent recovery. At this point further optimizations will lead to reduced emissions and costs at the same time.