Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes.     

The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

Rhodopsin, the prototypical G-protein-coupled receptor, which is densely packed in the disc membranes of rod outer segments, was proposed to function as a monomer. However, a growing body of evidence indicates dimerization and oligomerization of numerous G-protein-coupled receptors, and atomic force microscopy images revealed rows of rhodopsin dimers in murine disc membranes. In this work we demonstrate by electron microscopy of negatively stained samples, blue native- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, chemical crosslinking, and by proteolysis that native bovine rhodopsin exists mainly as dimers and higher oligomers. These results corroborate the recent findings from atomic force microscopy and molecular modeling on the supramolecular structure and packing arrangement of murine rhodopsin dimers.     

Projection maps of three members of the KdgM outer membrane protein family

We present the projection structures of the three outer membrane porins KdgM and KdgN from Erwinia chrysanthemi and NanC from Escherichia coli, based on 2D electron crystallography. A wide screening of 2D crystallization conditions yielded tubular crystals of a suitable size and quality to perform high-resolution electron microscopy. Data processing of untilted samples allowed us to separate the information of the two crystalline layers and resulted in projection maps to a resolution of up to 7 Å. All three proteins exhibit a similar putative β-barrel structure and the three crystal forms have the same symmetry. However, there are differences in the packing arrangements of the monomers as well as the densities of the projections. To interpret these projections, secondary structure prediction was performed using β-barrel specific prediction algorithms. The predicted transmembrane β-barrels have a high similarity in the arrangement of the putative β-strands and the loops, but do not match those of OmpG, a related protein porin whose structure was solved.     

Enzymatic preparation of 1,6-anhydro-muropeptides by immobilized murein hydrolases from Escherichia coli fused to staphylococcal protein A

Summary In order to produce biologically active 1,6-anhydro-muropeptides in large amounts by enzymatic degradation of isolated bacterial murein polymer highly specific periplasmic murein-metabolizing enzymes from Escherichia coli are made available. The genes slt, dacB, and mepA, encoding the soluble lytic transglycosylase (Slt), the penicillin-sensitive DD-endopeptidase (PBP4), and the penicillin-insensitive murein endopeptidase A (MepA), were independently fused to the N-terminal encoding sequence of staphylococcal protein A (SpA) under control of the temperature-inducible phage p _R promoter. The SpA fusion proteins were stably over-produced at high levels in E. coli upon temperature induction at 42°C and account for 3% (5 mg SpASlt/l culture), 3% (5 mg SpAPBP4/l culture), and 0.3% (0.5 mg SpAMepA/l culture) of total protein. The SpA fusion proteins, immobilized on IgG Sepharose, are proteolytically sensitive, in vitro, resulting in complete degradation of the SpA portion of the fusion proteins and release of the murein hydrolases in intact and enzymatically active form into the supernatant. Proteolytic degradation could be prevented by p-hydroxymercuribenzoic acid (PHMB) or ethylenediaminetetraacetate (EDTA) suggesting the involvement of the periplasmic protease Pi from E. coli. The immobilized fusion proteins were enzymatically active and could be used for the batch production of biologically active 1,6-anhydro-muropeptides, which were successively separated on HPLC. Isolated murein polymer was degraded quantitatively to monomeric 1,6-anhydro-muropeptides when immunoglobulin G (IgG)-SpASlt was used in combination with IgG-SpAMepA. A combination of IgG-SpASlt with IgG-SpAPBP4 left the 1,6-anhydro-dimers and oligomers being cross-linked via an LD-peptide bond (m-DAP-m-DAP) uncleaved. Correspondence to: W. Keck     

Structural insights into the secretin PulD and its trypsin-resistant core

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane beta-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.     

Temperature responses of three Fibrocapsa japonica strains (Raphidophyceae) from different climate regions

The harmful bloom alga Fibrocapsa japonica has a worldwide distributionin temperate regions and is occasionally responsible for massmortality of fish. Little is known about requirements for optimalgrowth and survival of this species, especially about temperatureconstraints that define natural distribution. Therefore, westudied thermal traits in three Fibrocapsa strains from differentclimate regions. All strains were eurythermal and viable between4 and 32°C, explaining their presence in temperate regions.Some differences in temperature response among the strains wereobserved, not only for growth rate but also for biovolume andnet production. The implication of the observed responses wasevaluated by translating growth performance of strains in thelaboratory to potential performance in the natural habitats.Only the Japanese strain seemed to be well adapted to its environment,while the New Zealand strain exhibited growth and survival overa much broader temperature range, despite the small temperaturefluctuations in its habitat. Interestingly, the German WaddenSea strain encounters lethal temperatures in winter and musthave a resting stage, able to survive temperatures <4°C,to explain its occurrence in this region. However, in general,the responses of the three F. japonica strains in culture werein good agreement with the observed seasonal occurrence in thefield.     

A genomic reservoir for Tnfrsf genes is developmentally regulated and imprinted in the mouse

Tumor necrosis factor receptor superfamily is composed of at least 26 members in the mouse, three of which exist as a cluster within the imprinted Kcnq1 domain on chromosome 7. Tnfrsf22, 23 and 26 contain typical cystein-rich domains and Tnfrsf22 and 23 can bind ligands but have no signaling capacity. Thus, they are assumed to be decoy receptors. The developmental expression profile of these genes is unknown and knowledge of their imprinting patterns is incomplete and controversial. We found that all three genes are expressed during mouse embryonic development, and that they have a strong maternal bias, indicating that they may be affected by the KvDMR, the Kcnq1 imprinting control region. We found expression of an antisense non-coding RNA, {"type":"entrez-nucleotide","attrs":{"text":"AK155734","term_id":"74212930","term_text":"AK155734"}}AK155734, in embryos and some neonatal tissues. This RNA overlaps the Tnfrsf22 and possibly the Tnfrsf23 coding regions and is also expressed with a maternal bias. We were interested in exploring the evolutionary origins of the three Tnfrsf genes, because they are absent in the orthologous human Kcnq1 domain. To determine whether the genes were deleted from humans or acquired in the rodent lineage, we performed phylogenetic analyses. Our data suggest that TNFRSF sequences were duplicated and/or degenerated or eliminated from the KCNQ1 region several times during the evolution of mammals. In humans, multiple mutations (point mutations and/or deletions) have accumulated on the ancestral TNFRSF, leaving a single short non-functional sequence.     

Arylalkylamine N-acetyltransferase gene expression in retina and pineal gland of rats under various photoperiods

The present study examines how the circadian oscillators in the retina and the suprachiasmatic nucleus (SCN) respond to changes in photoperiod. Arylalkylamine N-acetyltransferase (aa-nat) gene expression studied by quantitative RT-PCR revealed that in adult Sprague-Dawley rats kept under different light-dark (LD) cycles for two weeks the temporal pattern of AA-NAT mRNA expression was identical in retina and pineal gland. In both tissues, the time span between the onset of darkness and the nocturnal rise in AA-NAT mRNA expression was 3 h under LD 20:4, 6 h under LD 12:12, and 15 h under LD 4:20. As aa-nat expression in the pineal gland is regulated by the circadian oscillator in SCN, the results suggest that the photoperiodic differences accompanying the seasons of the year are imprinted in more than one oscillator and that this may accentuate the important message regarding 'time of year.'