A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans.

The human pathogenic yeast Candida albicans and a number of other Candida species translate the standard leucine CUG codon as serine. This is the latest addition to an increasing number of alterations to the standard genetic code which invalidate the theory that the code is frozen and universal. The unexpected finding that some organisms evolved alternative genetic codes raises two important questions: how have these alternative codes evolved and what evolutionary advantages could they create to allow for their selection? To address these questions in the context of serine CUG translation in C.albicans, we have searched for unique structural features in seryl-tRNA(CAG), which translates the leucine CUG codon as serine, and attempted to reconstruct the early stages of this genetic code switch in the closely related yeast species Saccharomyces cerevisiae. We show that a purine at position 33 (G33) in the C.albicans Ser-tRNA(CAG) anticodon loop, which replaces a conserved pyrimidine found in all other tRNAs, is a key structural element in the reassignment of the CUG codon from leucine to serine in that it decreases the decoding efficiency of the tRNA, thereby allowing cells to survive low level serine CUG translation. Expression of this tRNA in S.cerevisiae induces the stress response which allows cells to acquire thermotolerance. We argue that acquisition of thermotolerance may represent a positive selection for this genetic code change by allowing yeasts to adapt to sudden changes in environmental conditions and therefore colonize new ecological niches.     

Aspects of Gondwana paleobotany: gymnosperms of the Paleozoic—Mesozoic transition

During the late Paleozoic and early Mesozoic the Gondwana Supercontinent underwent dramatic geographic and climatic changes. Geologic and biologic factors concurrently played an important role modelling the vegetation of that time. The gymnospermic component of plant assemblages shows significant variations in composition and in the distribution of different taxa. Analysis of the assemblages shows that some plant groups dominated the scenario, such as the pteridosperms, glossopterids, corystosperms and, to a lesser degree, cordaites and conifers. Ginkgophytes, bennettites or cycads were less important in the Paleozoic but their numbers increased in the Triassic. Paleozoic assemblages were extensively dominated by glossopterids that became extinct in the earliest Mesozoic. Pteridosperms crossed the P—M barrier and became dominant during the Triassic, at a time when corystosperms evolved and radiated. Other groups became extinct in the Paleozoic, namely dicranophylls and cordaites. Conifers were represented by different families, restricted either to the Paleozoic or the Mesozoic. They were not conspicuous in the analysed assemblages. In some areas of Gondwana, taxa of the Euramerican alliance are present through a migrational mechanism that occurred during continental displacements which produced global climatic changes. Recent studies have shown that there are far more common elements between Euramerica and Gondwana than suspected up to now. These elements find their distribution especially in the western part of Gondwana (Africa—South America).     

Enrichment of mixed cultures capable of aerobic degradation of 1,2-dibromoethane.

1,2-dibromoethane (DBE) is a common environmental contaminant; it is potentially carcinogenic and has been detected in soil and groundwater supplies. Most of the biodegradation studies to date have been performed under anaerobic conditions or in the context of soil remediation, where the pollutant concentration was in the parts per billion range. In this work a mixed bacterial culture capable of complete aerobic mineralization of concentrations of DBE up to 1 g liter(-1) under well-controlled laboratory conditions was enriched. In order to verify biodegradation, formation of biodegradation products as well as the disappearance of DBE from the biological medium were measured. Complete mineralization was verified by measuring stoichiometric release of the biodegradation products. This mixed culture was found to be capable of degrading other halogenated compounds, including bromoethanol, the degradation of which has not been reported previously.     

Enzymatic hydrolysis of wood: an engineering assessment

Eucalyptus wood samples were delignified with HCl-catalysed acetic acid solutions under selected experimental conditions and treated with NaClO solutions. The solid residues obtained were employed as substrates for enzymatic hydrolysis. The NaClO concentration used in the pretreatment step, the liquor/solid ratio and the enzyme/substrate ratio were considered as operational variables. The experimental data allowed the development of generalized kinetic models which provided the necessary information for design calculations. The operational conditions were compared from an engineering viewpoint on the basis of economic estimates. Optimum conditions were established from these estimates.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Extraction and biodegradation of a toxic volatile organic compound (1,2-dichloroethane) from waste-water in a membrane bioreactor

An extractive membrane bioreactor has been used to treat a synthetic waste-water containing a toxic volatile organic compound, 1,2-dichloroethane (DCE). Biofilms growing on the surface of the membrane tubes biodegrade DCE while avoiding direct contact between the DCE and the aerating gas. This reduces air stripping by more than an order of magnitude (from 30–35% of the DCE entering the system to less than 1%) relative to conventional aerated bioreactors. Over 99% removal of DCE from a waste-water containing 1600 mg l^–1 of DCE was achieved at waste-water residence times of 0.75 h. Biodegradation was verified as the removal mechanism through measurements of CO₂ and chloride ion evolution in the bioreactor. No DCE was detected in the biomedium over the operating period. The diffusion-reaction phenomena occurring in the biofilm have been described by a mathematical model, which provides calculated solutions that support the experimental results by predicting that all DCE is biodegraded within the biofilm. Experimentally, however, the rate of DCE degradation in the biofilm was found to be independent of O₂ concentration, while the model predictions point to O₂ being limiting.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.