Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell.

The development of the Drosophila R7 photoreceptor cell is determined by a specific inductive interaction between the R8 photoreceptor cell and a single neighboring precursor cell. This process is mediated by bride of sevenless (boss), a cell-surface bound ligand, and the sevenless (sev) tyrosine kinase receptor. The boss ligand is expressed specifically on the surface of the R8 cell, whereas the sev receptor is expressed on 5 cells contacting the developing R8 cell and other cells not in contact with R8. By altering the spatial and temporal expression of boss, we demonstrate that sev-expressing cells that do not contact R8 can assume an R7 cell fate. By contrast, the sev-expressing precursor cells to the R1-R6 photoreceptor cells that do contact R8 are nonresponsive to the inductive cue. Using the rough and Nspl mutations, we demonstrate that an early commitment to an R1-R6 cell fate blocks the pathway of sev activation in these cells.     

Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC₅₀: 1 μM) led to the most potent compound 27 (IC₅₀: 0.038 μM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.     

Expression of a bacterial carotene hydroxylase gene (crtZ) enhances UV tolerance in tobacco

Carotenoids are essential components of the photosynthetic apparatus involved in plant photoprotection. To investigate the protective role of zeaxanthin under high light and UV stress we have increased the capacity for its biosynthesis in tobacco plants (Nicotiana tabacum L. cv. Samsun) by transformation with a heterologous carotenoid gene encoding -carotene hydroxylase (crtZ) from Erwinia uredovora under constitutive promoter control. This enzyme is responsible for the conversion of -carotene into zeaxanthin. Although the total pigment content of the transgenics was similar to control plants, the transformants synthesized zeaxanthin more rapidly and in larger quantities than controls upon transfer to high-intensity white light. Low-light-adapted tobacco plants were shown to be susceptible to UV exposure and therefore chosen for comparative analysis of wild-type and transgenics. Overall effects of UV irradiation were studied by measuring bioproductivity and pigment content. The UV exposed transformed plants maintained a higher biomass and a greater amount of photosynthetic pigments than controls. For revelation of direct effects, photosynthesis, pigment composition and chlorophyll fluorescence were examined immediately after UV treatment. Low-light-adapted plants of the crtZ transgenics showed less reduction in photosynthetic oxygen evolution and had higher chlorophyll fluorescence levels in comparison to control plants. After 1 h of high-light pre-illumination and subsequent UV exposure a greater amount of xanthophyll cycle pigments was retained in the transformants. In addition, the transgenic plants suffered less lipid peroxidation than the wild-type after treatment with the singlet-oxygen generator rose bengal. Our results indicate that an enhancement of zeaxanthin formation in the presence of a functional xanthophyll cycle contributes to UV stress protection and prevention of UV damage.     

Metabolism of 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose in yeast and chick-embryo cells.

2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose.     

Chiral aspects of metabolism of antiinflammatory drug flobufen in human hepatocytes

The metabolism of the nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in primary cultures of human hepatocytes prepared by two-step collagenase perfusion of livers from four donors. Racemic flobufen or its individual enantiomers, R-(+)- and S-(-)-flobufen were used as substrates. Aliquots of culture medium were collected during 24-h incubation. The time-dependent disappearance of flobufen enantiomers and the formation of metabolites (stereoisomers of dihydroflobufen (DHF)) in hepatocytes were measured by chiral HPLC. The reduction of flobufen in human hepatocytes was stereoselective ((+)-R-flobufen was preferentially metabolized) and stereospecific ((2R;4S)-DHF and (2S;4S)-DHF stereoisomers were mostly formed). Although the structure of flobufen is different from the profens (2-arylpropionates), flobufen undergoes chiral inversion in human hepatocytes. The inversion of R-(+)-flobufen to S-(-)-flobufen predominates. The individual DHF stereoisomers were incubated in hepatocyte cultures and their biotransformation studied. The unidirectional chiral inversion of (2S;4S)-DHF to (2R;4S)-DHF and (2R;4R)-DHF to (2S;4R)-DHF was observed. Stereoselective oxidation of the DHFs to flobufen was also detected. Thus, flobufen metabolism in primary cultures of human hepatocytes is much more complicated (via chiral inversion and DHF re-oxidation) than was presumed from a preliminary achiral point of view.     

The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

Multivariate statistical approach to the temporal and spatial patterns of selected bioindicators observed in the North Sea during the years 1995–1997

A comprehensive database, containing biological and chemical information, collected in the framework of the bilateral interdisciplinary MARS project (”biological indicators of natural and man-made changes in marine and coastal waters”) during the years 1995–1997 in the coastal environment of the North Sea, was subjected to a multivariate statistical evaluation. The MARS project was designated to combine a variety of approaches and to develop a set of methods for the employment of biological indicators in pollution monitoring and environmental quality assessment. In total, nine ship cruises to four coastal sampling sites were conducted; 765 fish and 384 mussel samples were analysed for biological and chemical parameters. Additional information on the chemical background at the sampling sites was derived from sediment samples, collected at each of the four sampling sites. Based on the available chemical data in sediments and black mussel (Mytilus edulis) a pollution gradient between the selected sites, was established. The chemical body burden of flounder (Platichthys flesus) from these sites, though, did not reflect this gradient equally clear. In contrast, the biological information derived from measurements in fish samples displayed significant a regional as well as a temporal pattern. A multivariate bioindicator data matrix was evaluated employing a factor analysis model to identify relations between selected biological indicators, and to improve the understanding of a regional and temporal component in the parameter response. In a second approach, applying the k-means algorithm on the data matrix, two significantly different clusters of samples, characterised by the current health status of the fish, were extracted. Using this classification a temporal, and in the second order, a less pronounced spatial effect was evident. In particular, during July 1996, a clear sign of deteriorating environmental conditions was extracted from the biological data matrix. Received: 20 June 1999 / Received in revised form: 8 November 1999 / Accepted: 8 November 1999     

Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria

Efficient protein secretion is very important in biotechnology as it provides active and stable enzymes, which are an essential prerequisite for successful biocatalysis. Therefore, optimizing enzyme-producing bacterial strains is a major challenge in the field of biotechnology and protein production. In this study, the Gram-positive model bacterium Bacillus subtilis was optimized for heterologous protein secretion using a novel approach. Two lipolytic enzymes, cutinase from Fusarium solani pisi and a cytoplasmatic esterase of metagenomic origin, were chosen as reporters for heterologous protein secretion. In a systematic screening approach, all naturally occurring (non-lipoprotein) Sec-type signal peptides (SPs) from B. subtilis were characterized for their potential in heterologous protein secretion. Surprisingly, optimal SPs in cutinase secretion were inefficient in esterase secretion and vice versa, indicating the importance of an optimal fit between the SP and the respective mature part of the desired secretion target proteins. These results highlight the need for individually optimal signal peptides for every heterologous secretion target. Therefore, the SP library generated in this study represents a powerful tool for secretion optimization in Gram-positive expression hosts.     

Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.