Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.)

The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2--galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.     

Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells

Abstract. Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the 'selected' cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le^y, Le^x, sialosyl-Le^a, precursor of Thomsen Friedenreich antigen (T_n), but not Thomsen-Friedenreich antigen and sialosyl-T_n]. Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2⁺ cells develop from the basal cell layer of the mammary gland.     

Anaerobic degradation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by a marine Desulfobacterium strain

Dimethylsulfoniopropionate, an osmolyte of marine algae, is thought to be the major precursor of dimethyl sulfide, which plays a dominant role in biogenic sulfur emission. The marine sulfate-reducing bacterium Desulfobacterium strain PM4 was found to degrade dimethylsulfoniopropionate to 3-S-methylmercaptopropionate. The oxidation of one of the methyl groups of dimethylsulfoniopropionate was coupled to the reduction of sulfate; this process is similar to the degradation betaine to dimethylglycine which was described earlier for the same strain. Desulfobacterium PM4 is the first example of an anaerobic marine bacterium that is able to demethylate dimethylsulfoniopropionate.Abbreviations DMSP dimethylsulfoniopropionate - DMS dimethyl sulfide - MMPA 3-S-methylmercaptopropionate     

Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway

The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.Cell extracts of pyruvate-grown P.furiosus contained the following enzyme activities: phosphoenolpyruvate synthetase (0.025 U/mg, 50 °C), enolase (0.9 U/mg, 80 °C), phosphoglycerate mutase (0.13 U/mg, 55 °C), phosphoglycerate kinase (0.01 U/mg, 50 °C), glyceraldehyde-3-phosphate dehydrogenase reducing either NADP⁺ or NAD⁺ (NADP⁺: 0.019 U/mg, NAD⁺: 0.009 U/mg; 50 °C), triosephosphate isomerase (1.4 U/mg, 50 °C), fructose-1,6-bisphosphate aldolase (0.0045 U/mg, 55 °C), fructose-1,6-bisphosphate phosphatase (0.026 U/mg, 75 °C), and glucose-6-phosphate isomerase (0.22 U/mg, 50 °C). Kinetic properties (V _max values and apparent K _m values) of the enzymes indicate that they operate in the direction of sugar synthesis. The specific enzyme activities of phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADP⁺-reducing) and fructose-1,6-bisphosphate phosphatase in pyruvate-grown P. furiosus were by a factor of 3, 10 and 4, respectively, higher as compared to maltose-grown cells suggesting that these enzymes are induced under conditions of gluconeogenesis. Furthermore, cell extracts contained ferredoxin: NADP⁺ oxidoreductase (0.023 U/mg, 60 °C); phosphoenolpyruvate carboxylase (0.018 U/mg, 50 °C) acts as an anaplerotic enzyme.Thus, in P. furiosus sugar formation from pyruvate involves reactions of the Embden-Meyerhof pathway, whereas sugar degradation to pyruvate proceeds via a modified non-phosphorylated Entner-Doudoroff pathway.     

Root Morphology and Nitrogen Uptake of Maize Simultaneously Supplied with Ammonium and Nitrate in a Split-root System

We studied the response of maize (Zea mays L. cv. Anjou 256)to a simultaneous, but separated supply of ammonium and nitrate(localized supply, LS). A split-root system was used to supplyhalf of the roots with ammonium and the other half with nitrate.A homogeneously distributed supply of both nitrogen forms (HS)was the control treatment. Seedlings were grown for 12 d fromthe two-leaf to the three-leaf stage in hydroponics at threepH levels (4, 5·5 and 7). The total N concentration was3 mol m^-3. The split-root system was established by removingthe seminal root system and using only four nodal roots perplant. Total root length and root surface area were recordedautomatically with a modified Delta- T area meter. Other morphologicalroot traits (such as main axis length and diameter, number,density, and length of laterals) were recorded manually. Uptakeof ammonium and nitrate was measured by the depletion of thenutrient solution. As compared with LS, HS was superior in shootand root DM, total root length and root surface area, ammoniumand nitrate uptake and shoot nitrogen concentration, irrespectiveof pH level. This indicates that, also under field conditions,mixed ammonium and nitrate fertilization is only beneficialto plant growth if both N forms are evenly distributed in thesoil. At both HS and LS, ascending pH increased the ammonium:nitrateuptake ratio. At LS, declining pH induced a considerable shiftin the distribution of root DM, root length, and root surfacearea the nitrate-fed compartment.Copyright 1993, 1999 AcademicPress Maize, Zea may L., ammonium, nitrate, pH, root morphology, split-root     

The 11p15.5 ribonucleotide reductase M1 subunit locus is not imprinted in Wilms' tumour and hepatoblastoma

We have utilized the polymerase chain reaction (PCR) to amplify a transcribed Taq1 polymorphism in the ribonucleotide reductase M1 subunit (RRM1) gene at chromosome 11p15.5, to investigate whether this locus is subjected to imprinting in embryonal tumours. The polymorphism was amplified from cDNA from 6 Wilms' tumours, one hepatoblastoma and corresponding samples of adjacent kidney or liver from individuals who were constitutionally heterozygous for the polymorphism. Taq1 digestion of PCR products revealed that both alleles were transcribed in all samples where both were present at the genomic level, indicating that the RRM1 locus is not subjected to imprinting in Wilms' tumour or hepatoblastoma.     

The human complement component C8B gene: structure and phylogenetic relationship

The eighth component of human complement (C8) is a serum protein that consists of three chains (, and ), encoded by three separate genes, viz., C8A, C8B, and C8G. In serum, the -subunit is non-covalently bound to the disulfide-linked - subunit. Using a full-length C8 cDNA probe, we isolated several clones from human genomic DNA libraries. Four clones covering the complete cDNA sequence were characterized by TaqI restriction mapping and were shotgun subcloned into M13. C8-cDNA-positive clones were partially sequenced to characterize the 12 exons of the gene with sizes from 69 to 347 bp. All intron-exon junctions followed the GT-AG rule. By using polymerase chain reaction (PCR) primers located in the adjacent intron sequences, all 12 exons of the C8B gene could be amplified from genomic DNA. All fragments showed the expected sizes. The sizes of eight introns could be determined by using primer pairs that amplified two exons and the enclosed intron, and by restriction mapping. These analyses and the insert sizes of the genomic clones indicate that the C8B gene has a total size of approximately 40 kb. The polymorphic TaqI site of the C8B gene localized in intron 11 could be demonstrated by direct restriction fragment analysis of a PCR fragment containing exons 11 and 12, and the enclosed intron 11. Homology comparison of the C8B gene with C8A and C9 on the basis of the exon structure confirmed the ancestral relationship known from the protein level.