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.'     

Association between TAS2R38 gene polymorphisms and colorectal cancer risk: a case-control study in two independent populations of Caucasian origin

Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99–1.67; P_value = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06–1.75; P_value = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12–1.61; P_value = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin.     

Ox liver glutamate dehydrogenase. The role of lysine-126 reappraised in the light of studies of inhibition and inactivation by pyridoxal 5''-phosphate.

The time-course of inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5'-phosphate was studied in the presence of varied amounts of 2-oxoglutarate or NADH. Pseudo-first-order analysis reveals that the protection by both these compounds is competitive with respect to the chemical modifier. The competition is only partial, however: saturation with either NADH or 2-oxoglutarate decreases the rate constant for inactivation to a finite minimum and not to zero. Similarly, the plot of activity at equilibrium as a function of the concentration of the protecting substrate or coenzyme reveals that neither NADH nor 2-oxoglutarate protects completely against inactivation. In initial-rate experiments, pyridoxal 5'-phosphate, used as an instantaneous inhibitor rather than a long-term inactivator, displayed non-competitive inhibition with respect to both 2-oxoglutarate and NADH. These results clearly indicate that, although there is mutual hindrance between the binding to the enzyme of pyridoxal 5'-phosphate, on the one hand, and 2-oxoglutarate or NADH on the other, binding is not mutually exclusive. These findings are discussed in terms of the two-step mechanism for inactivation by pyridoxal 5'-phosphate. It is concluded that lysine-126 cannot be solely responsible for binding either the substrate or the coenzyme, but could be essential for the catalytic step.     

Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography.

Molecular water channels (aquaporins) allow living cells to adapt to osmotic variations by rapid and specific diffusion of water molecules. Aquaporins are present in animals, plants, algae, fungi and bacteria. Here we present an electron microscopic analysis of the most ancient water channel described so far: the aquaporin Z (AqpZ) of Escherichia coli. A recombinant AqpZ with a poly(histidine) tag at the N terminus has been constructed, overexpressed and purified to homogeneity. Solubilized with octylglucoside, the purified AqpZ remains associated as a homotetramer, and assembles into highly ordered two-dimensional tetragonal crystals with unit cell dimensions a = b = 95 A, gamma = 90 degrees when reconstituted by dialysis in the presence of lipids. Three-dimensional reconstruction of negatively stained lattices revealed the p42(1)2 packing arrangement that is also observed with the human erythrocyte water channel (AQP1). The 8 A projection map of the AqpZ tetramer in frozen hydrated samples is similar to that of AQP1, consistent with the high sequence homology between these proteins.     

Multiple Mißbildungen bei einem Mädchen mit dem Karyotypus 46, XY, 17q+

Summary In a girl with multiple malformations (Pierre Robin syndrome, abortive form of dysostosis cleidocranialis, and other deformities) a spontaneous duplication-deficiency as well as the XY sex chromosomes were found. Detailed cytogenetic analysis revealed a deficiency of the distal half of the long arm of a chromosome 17. The duplication involves a region of the size of the long arm of chromosome 18. The combination of malformations as well as the autoradiographic pattern does not exclude the possibility that indeed a region of the long arm of chromosome 18 is involved.

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Wesentliche Teile der vorliegenden Arbeit werden von Herrn Dieter Bombel als Dissertation der Medizinischen Fakultät der Universität Freiburg i. Br. vorgelegt.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.