Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice

The large scale production of recombinant collagen for use in biomaterials requires an efficient expression system capable of processing a large (>400Kd) multisubunit protein requiring post-translational modifications. To investigate whether the mammary gland of transgenic animals fulfills these requirements, transgenic mice were generated containing the S1-casein mammary gland-specific promoter operatively linked to 37Kb of the human 1(I) procollagen structural gene and 3 flanking region. The frequency of transgenic lines established was 12%. High levels of soluble triple helical homotrimeric [(1)₃] type I procollagen were detected (up to 8mg/ml) exclusively in the milk of six out of 9 lines of lactating transgenic mice. The transgene-derived human procollagen chains underwent efficient assembly into a triple helical structure. Although proline or lysine hydroxylation has never been described for any milk protein, procollagen was detected with these post-translational modifications. The procollagen was stable in mil; minimal degradation was observed. These results show that the mammary gland is capable of expressing a large procollagen gene construct, efficiently assembling the individual polypeptide chains into a stable triple helix, and secreting the intact molecule into the milk.     

Crystals of acetylated SecB diffract to 2.3-A resolution

The molecular chaperone SecB is part of the protein translocation pathway in Escherichia coli. SecB was purified from an overproducing strain and crystallized, resulting in crystals diffracting to 2.3-A resolution. The analysis of electrospray ionization mass spectra of dissolved crystals of SecB indicated that we have crystallized an acetylated form of SecB. Sequence analysis suggests that the protein is fully acetylated at its N-terminus in vivo, indicating that potential deacetylation is artificially introduced by purification methods. The high degree of acetylation that we observed might account for the fact that the crystals obtained as described in this study diffract to higher resolution than those in previously reported trials.     

A small, cysteine-rich protein secreted by Fusarium oxysporum during colonization of xylem vessels is required for I-3-mediated resistance in tomato

A 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I-3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I-3 line. Conversely, deletion of the SIX1 gene in a wild-type strain results in breaking of I-3-mediated resistance. These results suggest that I-3-mediated resistance is based on recognition of Six1 secreted in xylem vessels.     

Structural characterization of Botryosphaeran: a (1-->3;1-->6)-beta-D-glucan produced by the ascomyceteous fungus,Botryosphaeria sp

The exopolysaccharide, Botryosphaeran, produced by the ligninolytic, ascomyceteous fungus Botryosphaeria sp., was isolated from the extracellular fluid by precipitation with ethanol, and purified by gel permeation chromatography to yield a carbohydrate-rich fraction (96%) composed mainly of glucose (98%). Infra-red and 13C NMR spectroscopy showed that all the glucosidic linkages were in the beta-configuration. Data from methylation analysis and Smith degradation indicated that Botryosphaeran was a (1-->3)-beta-D-glucan with approx 22% side branching at C-6. The products obtained from partial acid hydrolysis demonstrated that the side branches consisted of single (1-->6)-beta-linked glucosyl, and (1-->6)-beta-linked gentiobiosyl residues.     

A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins

The coding sequence of a major xylem sap protein of tomato was identified with the aid of mass spectrometry. The protein, XSP10, represents a novel family of extracellular plant proteins with structural similarity to plant lipid transfer proteins. The XSP10 gene is constitutively expressed in roots and lower stems. The decline of XSP10 protein levels in tomato infected with a fungal vascular pathogen may reflect breakdown or modification by the pathogen.     

Distinct epithelial responses in experimental colitis: implications for ion uptake and mucosal protection

In the present study, we aimed to investigate enterocyte- and goblet cell-specific functions during the different phases of acute colitis induced with dextran sulfate sodium (DSS). Rats were treated with DSS for 7 days, followed by a 7-day recovery period. Colonic tissue was excised on days 2 (onset of disease), 7 (active disease), and 14 (regenerative phase). Enterocyte functions were studied by the expression of carbonic anhydrases (CAs), sodium/hydrogen exchangers (NHEs) and intestinal fatty acid-binding protein (iFABP) and by alkaline phosphatase (AP) activity. The expression and secretion of the mucin Muc2 and trefoil factor family peptide-3 (TFF3) were used as parameters for goblet cell function. DSS induced a downregulation of the CAs, NHEs, and iFABP in some normal-appearing surface enterocytes and in most of the flattened-surface enterocytes during disease onset and active disease. During the regenerative phase most enterocytes expressed these genes again. Quantitative analysis revealed a significant decrease in CAs, NHEs, and iFABP expression levels during onset and active disease. During the regenerative phase, the expression levels of the CAs were restored, whereas the expression levels of the NHEs and iFABP remained decreased. In contrast, enterocyte-specific AP activity was maintained in normal and flattened enterocytes during DSS-induced colitis. Goblet cells continued to express MUC2 and TFF3 during and after DSS treatment. Moreover, Muc2 and TFF3 expression and secretion levels were maintained or even increased during each of the DSS-induced disease phases. In conclusion, DSS-induced colitis was associated with decreased expression of CAs, NHEs, and iFABP. The loss of these genes possibly accounts for some of the pathology seen in colitis. The maintenance or upregulation of Muc2 and TFF3 synthesis and secretion levels implies that goblet cells at least maintain their epithelial defense and repair capacity during acute inflammation induced by DSS.     

High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines

A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files. Data quality was significantly improved by (i) restriction digestion of the adaptor-ligation products to reduce trivial sequences caused by co-amplification of fragments derived from the free plasmid, and (ii) the design of the adaptor primers for the second amplification step to enhance selective generation of single PCR fragments, even from lines with multiple T-DNA insertions. Gel-purification was avoided by including these steps, the number of amplification reactions per line was reduced from four to three, and the percentage of lines that yielded at least one FST was increased from 66% to 86%. More than 58,000 FSTs have been submitted to GenBank and are available at http://www.mpiz-koeln.mpg.de/GABI-Kat/.     

Selective sparing of goblet cells and paneth cells in the intestine of methotrexate-treated rats

Proliferation, differentiation, and cell death were studied in small intestinal and colonic epithelia of rats after treatment with methotrexate. Days 1-2 after treatment were characterized by decreased proliferation, increased apoptosis, and decreased numbers and depths of small intestinal crypts in a proximal-to-distal decreasing gradient along the small intestine. The remaining crypt epithelium appeared flattened, except for Paneth cells, in which lysozyme protein and mRNA expression was increased. Regeneration through increased proliferation during days 3-4 coincided with villus atrophy, showing decreased numbers of villus enterocytes and decreased expression of the enterocyte-specific genes sucrase-isomaltase and carbamoyl phosphate synthase I. Remarkably, goblet cells were spared at villus tips and remained functional, displaying Muc2 and trefoil factor 3 expression. On days 8-10, all parameters had returned to normal in the whole small intestine. No methotrexate-induced changes were seen in epithelial morphology, proliferation, apoptosis, Muc2, and TFF3 immunostaining in the colon. The observed small intestinal sparing of Paneth cells and goblet cells following exposure to methotrexate is likely to contribute to epithelial defense during increased vulnerability of the intestinal epithelium.     

Conformational changes in photosystem II supercomplexes upon removal of extrinsic subunits

Photosystem II is a multisubunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts. It consists of a large number of intrinsic membrane proteins involved in light-harvesting and electron-transfer processes and of a number of extrinsic proteins required to stabilize photosynthetic oxygen evolution. We studied the structure of dimeric supercomplexes of photosystem II and its associated light-harvesting antenna by electron microscopy and single-particle image analysis. Comparison of averaged projections from native complexes and complexes without extrinsic polypeptides indicates that the removal of 17 and 23 kDa extrinsic subunits induces a shift of about 1.2 nm in the position of the monomeric peripheral antenna protein CP29 toward the central part of the supercomplex. Removal of the 33 kDa extrinsic protein induces an inward shift of the strongly bound trimeric light-harvesting complex II (S-LHCII) of about 0.9 nm, and in addition destabilizes the monomer-monomer interactions in the central core dimer, leading to structural rearrangements of the core monomers. It is concluded that the extrinsic subunits keep the S-LHCII and CP29 subunits in proper positions at some distance from the central part of the photosystem II core dimer to ensure a directed transfer of excitation energy through the monomeric peripheral antenna proteins CP26 and CP29 and/or to maintain sequestered domains of inorganic cofactors required for oxygen evolution.