Specific Developmental Defects in Molluscs after Treatment with Retinoic Acid during Gastrulation

All-trans retinoic acid is known as a teratogen in vertebrate development. To study whether molluscan morphogenesis is sensitive to retinoic acid, the development of Lymnaea stagnalis, Physa fontinalis and Bithynia tentaculata was examined after treatment with retinoic acid. Low concentrations retinoic acid (10^–7M) specifically affected eye formation in each of these species. In Lymnaea , it was shown that 10^–6M retinoic acid resulted in a wider spectrum of deficiencies, including eye defects, arrested development and shell deformations. Pulse treatments revealed that embryos were most sensitive during gastrulation. Soon after gastrulation the embryos had lost their sensitivity to retinoic acid, which indicates that the observed defects do not result from a general non-specific toxic effect on cells. Even though molluscan development differs in numerous respects from vertebrate development, the present results suggest that molluscs share common features with vertebrates in morphogenetic processes that operate in early development.     

Mutations at position 1122 in the catalytic domain of the mouse ras-specific guanine nucleotide exchange factor CDC25 originate both loss-of-function and gain-of-function proteins

The role of two residues within the catalytic domain of CDC25^Mm, a mouse ras-specific guanine nucleotide exchange factor (GEF), was investigated by site-directed mutagenesis. The function of the mutant proteins was tested in vivo in both a Saccharomyces cerevisiae cdc25 complementation assay and in a mammalian fos-luciferase assay, and in in vitro assays on human and yeast Ras proteins. Mutants CDC25 and CDC25 were shown to be (partly) inactive proteins, similar to their yeast homologs. Mutant CDC25 showed higher nucleotide exchange activity than the wild type protein on the basis of both in vitro and in vivo assays. Thus, alanine and valine substitutions at position 1122 within the GEF catalytic domain originate mutations with opposite biological properties, indicating an important role for position 1122 in GEF function.     

Mixed culture biotechnology for bioenergy production

Mixed culture biotechnology (MCB) could become an attractive addition or alternative to traditional pure culture based biotechnology for the production of chemicals and/or bioenergy. On the basis of ecological selection principles, MCB-based processes can be established that generate a narrow product spectrum from a mixed substrate. Three example processes are briefly discussed in this paper: anaerobic digestion aimed at the production of methane-containing biogas, mixed culture fermentation for the production of solvents or biohydrogen, and a two-step process for the production of polyhydroxyalkanoates. These examples give an idea of the potential contribution of mixed culture biotechnology to sustainable production of bioenergy from waste.     

Analysis of microsatellite mutations in buccal cells from a case-control study for lung cancer

Exposure to tobacco carcinogens is the major cause of human lung cancer, but even heavy smokers have only about a 10% life-time risk of developing lung cancer. Currently used screening processes, based largely on age and exposure status, have proven to be of limited clinical utility in predicting cancer risk. More precise methods of assessing an individual's risk of developing lung cancer are needed. Because of their sensitivity to DNA damage, microsatellites are potentially useful for the assessment of somatic mutational load in normal cells. We assessed mutational load using hypermutable microsatellites in buccal cells obtained from lung carcinoma cases and controls to test if such a measure could be used to estimate lung cancer risk. There was no significant association between smoking status and mutation frequency with any of the markers tested. No significant association between case status and mutation frequency was observed. Age was significantly related to mutation frequency in the microsatellite marker D7S1482. These observations indicate that somatic mutational load, as measured using mutation frequency of microsatellites in buccal cells, increases with increasing age but that subjects who develop lung cancer have a similar mutational load as those who remain cancer free. This finding suggests that mutation frequency of microsatellite mutations in buccal cells may not be a promising biomarker for lung cancer risk.     

Genetic Diversity and Population Structure of Cucumber (Cucumis sativus L.)

Knowing the extent and structure of genetic variation in germplasm collections is essential for the conservation and utilization of biodiversity in cultivated plants. Cucumber is the fourth most important vegetable crop worldwide and is a model system for other Cucurbitaceae, a family that also includes melon, watermelon, pumpkin and squash. Previous isozyme studies revealed a low genetic diversity in cucumber, but detailed insights into the crop''s genetic structure and diversity are largely missing. We have fingerprinted 3,342 accessions from the Chinese, Dutch and U.S. cucumber collections with 23 highly polymorphic Simple Sequence Repeat (SSR) markers evenly distributed in the genome. The data reveal three distinct populations, largely corresponding to three geographic regions. Population 1 corresponds to germplasm from China, except for the unique semi-wild landraces found in Xishuangbanna in Southwest China and East Asia; population 2 to Europe, America, and Central and West Asia; and population 3 to India and Xishuangbanna. Admixtures were also detected, reflecting hybridization and migration events between the populations. The genetic background of the Indian germplasm is heterogeneous, indicating that the Indian cucumbers maintain a large proportion of the genetic diversity and that only a small fraction was introduced to other parts of the world. Subsequently, we defined a core collection consisting of 115 accessions and capturing over 77% of the SSR alleles. Insight into the genetic structure of cucumber will help developing appropriate conservation strategies and provides a basis for population-level genome sequencing in cucumber.     

Expression of agsA, one of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger, is induced in response to cell wall stress

1,3-alpha-D-Glucan is an important component of the cell wall of filamentous fungi. We have identified a family of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger. The agsA gene was sequenced and the predicted protein sequence indicated that the overall domain structure of 1,3-alpha-D-glucan synthases is conserved in fungi. Using RT-PCR and Northern blot analysis, we found that expression of the agsA gene and to a lesser extent also of agsE were induced in the presence of the cell wall stress-inducing compounds such as Calcofluor White (CFW), SDS, and caspofungin. Loss of agsA function did not result in an apparent phenotype under normal growth conditions but rendered the cells more sensitive to CFW. The induction of 1,3-alpha-D-glucan synthase-encoding genes in response to cell wall stress was not limited to A. niger, but was also observed in Penicillium chrysogenum. We propose that this response to cell wall stress commonly occurs in filamentous fungi.     

Bioanalysis and pharmacokinetics of the p38 MAPkinase inhibitor SB202190 in rats

We have developed a sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for the quantification of the p38 MAPkinase inhibitor SB202190 in serum, kidney homogenates and urine samples. Liquid-liquid extraction of SB202190 from the samples was performed using diethylether after adding a derivative of SB202190 as internal standard (I.S.). Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of acetonitrile-water-trifluoroacetic acid (30:70:0.1, v/v/v; pH 2.0). Both drug and I.S. were measured at 350 nm and eluted at 5.0 and 10.6 min, respectively. Peak-height ratios of the drug and the I.S. were used for the quantification of SB202190 from the different matrixes. The limit of quantitation of SB202190 in serum, kidney and urine were 0.25 microg/ml, 1 microg/g and 1 microg/ml, respectively. The average recoveries were 74, 75 and 92% in serum, kidney and urine, respectively. The intra- and inter-day precision (% CV) and accuracy (% bias) were below 15% for all concentrations. The method was successfully applied for a pharmacokinetic study of SB202190 in rats.     

Elongation factor Tu: a molecular switch in protein biosynthesis

Elongation factor Tu (EF-Tu), the most abundant protein in Escherichia coli, is a guanine nucleotide-binding protein that in the 'on' state acts as a carrier of amino acyl-tRNA to the ribosome. Our knowledge of this essential component of translation has brought substantial progress in the past decade thanks to the co-ordinated application of biochemical, physico-chemical and genetic methods. Crystallographic analysis at 2.6 A resolution and site-directed mutagenesis have revealed structural and functional similarities between the guanine nucleotide-binding domains of EF-Tu and human H-ras p21 protein. The regulation of the expression of the two EF-Tu-encoding genes in E. coli, particularly that of tufB, has been shown to involve diverse mechanisms. Several aspects of the functions of EF-Tu in the elongation cycle have been reinvestigated, leading to new insights. These studies have emphasized the manifold aspects of the mechanisms regulating the activity of EF-Tu in the bacterial cell.     

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.