Pore formation and function of phosphoporin PhoE of Escherichia coli are determined by the core sugar moiety of lipopolysaccharide

The lipid matrix of the outer membrane of Gram-negative bacteria is an asymmetric bilayer composed of a phospholipid inner leaflet and a lipopolysaccharide outer leaflet. Incorporated into this lipid matrix are, among other macromolecules, the porins, which have a sieve-like function for the transport or exclusion of hydrophilic substances. It is known that a reduced amount of porins is found in the outer membrane of rough mutants as compared with wild-type bacteria. This observation was discussed to be caused by a reduced number of insertion sites in the former. We performed electrical measurements on reconstituted planar bilayers composed of lipopolysaccharide on one side and a phospholipid mixture on the other side using lipopolysaccharide from various rough mutant strains of Salmonella enterica serovar Minnesota. We found that pore formation by PhoE trimers that were added to the phospholipid side of the bilayers increased with the increasing length of the lipopolysaccharide core sugar moiety. These results allow us to conclude that the length of the sugar moiety of lipopolysaccharide is the parameter governing pore formation and that no particular insertion sites are required. Furthermore, we found that the voltage gating of the porin channels is strongly dependent on the composition of the lipid matrix.     

Seasonal Changes in the Photosynthetic Efficiency of Thuja occidentalis (L.) and Chamaecyparis lawsonia (A. Murray bis.)

Abstract: Seasonal changes in the efficiency of charge separation in PSII were studied in Thuja occidentalis (L.) and Chamaecyparis lawsonia (A. Murray bis.). Maximum light-dependent charge separation decreased with decreasing temperatures in early winter in both species, but this was less drastic in Chamaecyparis than in Thuja. No positive relationship was seen between photoinhibition and irradiance. Rather, photoinhibition increased as photon flux densities decreased towards midwinter, and it decreased as photon flux densities increased towards spring. However, the decrease in maximum light-dependent charge separation was much stronger on the light-exposed upper surface of the twigs, where in Thuja visible browning occurred, than on the underside of the twigs. During spring, recovery of the photosynthetic efficiency and regreening were observed as both mean temperatures and irradiance increased. Transfer in midwinter of strongly photo-inhibited twigs of Thuja to temperatures close to 20 °C resulted in considerable recovery of PSII activity within several days when low light was also present. Recovery did not occur at temperatures close to freezing or at room temperature in darkness. An analysis of fluorescence quenching suggested photoprotective dissipation of excess radiation not only in the light harvesting antennae of PSII but also in the reaction centres. Reaction centre quenching appeared to be stronger in Thuja than in Chamaecyparis. PSI was fully active in twigs whether or not PSII was photoinhibited. The antioxidant ascorbate was almost fully reduced even in midwinter.     

Purification and characterization of the L-fucose transporter

L-Fucose is a monosaccharide present in low levels in the serum. It is, however, a common structural component of glycoproteins. L-Fucose is accumulated in eukaryotic cells by a specific, facilitative diffusion transport system which has been designated the fucose transporter. In this study, purification of the transporter from mouse brain was performed by detergent extraction followed by ion-exchange and reactive dye ligand column chromatography. Purification was followed using a transport assay into reconstituted liposomes. A 111-fold purification with 5% yield was achieved from the crude homogenate. The apparent molecular weight of the protein was 57 kDa. Transport was found to be saturable. The K(m) and V(max) values are estimated at 3 microM and 275 pmol/min/mg, respectively. The tissue distribution of fucose transport was examined in liver, kidney, heart, lung, spleen, brain, muscle, adipose, ovary, pancreas, and thymus. Some fucose transport was found in all tissues examined. Very low levels were observed in the liver relative to all other tissues examined. The only monosaccharide which could inhibit the uptake of L-[5,6-(3)H]fucose was fucose itself.     

Accumulation of the cell cycle regulators TP53 and CDKN1A (p21) in human fibroblasts after exposure to low- and high-LET radiation

The accumulation of the cell cycle regulators TP53 and CDKN1A (p21/CIP1/WAF1) was investigated after exposure to X rays and carbon ions (170 keV microm(-1)) and xenon, bismuth and uranium ions (8900-15,000 keV microm(-1)) in normal human fibroblasts. The influence of the overall dose and the LET of these radiation types was studied systematically and the kinetics of the cell response was followed up to 24 h after exposure. The accumulation of TP53 protein was dependent on the dose and the LET, and TP53 levels declined to lower levels for all radiation types within 24 h after exposure. CDKN1A levels increased and peaked at 3 to 6 h after exposure. The persisting level of this protein at 24 h was strongly dependent on the dose and the LET for X rays and carbon ions. The exposure to very high-LET ions (8900-15,000 keV microm(-1)) did not lead to a further increase in CDKN1A, suggesting a saturation effect for the induction of this protein. The cellular effects of elevated CDKN1A after particle irradiation are discussed.     

Structure-dependent effects of glucose-containing analogs of platelet activating factor (PAF) on membrane integrity

Synthetic choline-containing phospholipids comprise a new class of compounds with antineoplastic properties. We have investigated the effect of recently synthesized glucose-containing analogs of lysophosphatidylcholine (glyceroglucophospholipid, Glc-PC) and of lysoplatelet activating factor (Glc-PAF) and its C16, C14 and C12 derivatives (ET-16, ET-14, and ET-12) on proliferation of immortalized human keratinocyte (HaCaT) cells. The data were compared to the ability of the compounds to intercalate into phosphatidylserine liposomes and to form lesions in planar bilayer membranes. A correlation between bioactivity and membrane activity was found. The number of molecules that intercalated into phosphatidylserine liposomes depended on the chemical structure of the compounds and was in the order Glc-PAF approximately ET-16 approximately ET-14 > Glc-PC > ET-12. All compounds induced membrane lesions, and the lesion forming activity was in the same order. Similar activity rankings were found for the release of lactate dehydrogenase from HaCaT cells as a measure of lytic activity and for the influence on cell number as a measure of proliferation. In the latter test, however, proliferation was already inhibited at non-toxic concentrations. From these findings, it may be concluded that the intercalation of the compounds at toxic concentrations leads to the formation of membrane lesions and finally results in membrane rupture leading to cell death.     

Developmental motoneuron cell death and neurotrophic factors

During the development of higher vertebrates, motoneurons are generated in excess. In the lumbar spinal cord of the developing rat, about 6000 motoneurons are present at embryonic day 14. These neurons grow out axons which make contact with their target tissue, the skeletal muscle, and about 50% of the motoneurons are lost during a critical period from embryonic day 14 until postnatal day 3. This process, which is called physiological motoneuron cell death, has been the focus of research aiming to identify neurotrophic factors which regulate motoneuron survival during this developmental period. Motoneuron cell death can also be observed in vitro when the motoneurons are isolated from the embryonic avian or rodent spinal cord. These isolated motoneurons and other types of primary neurons have been a useful tool for studying basic mechanisms underlying neuronal degeneration during development and under pathophysiological conditions in neurodegenerative disorders. Accumulating evidence from such studies suggests that some specific requirements of motoneurons for survival and proper function may change during development. The focus of this review is a synopsis of recent data on such specific mechanisms.     

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein–lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.     

cRNA target preparation for microarrays: comparison of gene expression profiles generated with different amplification procedures

Microarray technology has become a standard tool for generation of gene expression profiles to explore human disease processes. Being able to start from minute amounts of RNA extends the fields of application to core needle biopsies, laser capture microdissected cells, and flow-sorted cells. Several RNA amplification methods have been developed, but no extensive comparability and concordance studies of gene expression profiles are available. Different amplification methods may produce differences in gene expression patterns. Therefore, we compared profiles processed by a standard microarray protocol with three different types of RNA amplification: (i) two rounds of linear target amplification, (ii) random amplification, and (iii) amplification based on a template switching mechanism. The latter two methods accomplish target amplification in a nonlinear way using PCR technology. Starting from as little as 50 ng of total RNA, the yield of labeled cRNA was sufficient for hybridization to Affymetrix HG-U133A GeneChip array using the respective methods. Replicate experiments were highly reproducible for each method. In comparison with the standard protocol, all three approaches are less sensitive and introduced a minor but clearly detectable bias of the detection call. In conclusion, the three amplification protocols used are applicable for GeneChip analysis of small tissue samples.     

The Xenopus TACC homologue, maskin, functions in mitotic spindle assembly

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a approximately 200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to "rescue" experiments. Defects in the "early" step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the "late" step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.