Stimulation of the diadinoxanthin cycle by UV-B radiation in the diatom Phaeodactylum tricornutum

In this study we show that the diadinoxanthin cycle in the diatom Phaeodactylum tricornutum is stimulated by mild UV-B radiation. High steady state concentrations of diatoxanthin established during a period of strong actinic illumination with white light (300 mol photons m-2 s-1 PAR) are further increased if weak UV-B (3 mol photons m-2 s-1) is additionally applied. Short term increases in the diatoxanthin concentration caused by UV-B strongly correlate with a stoichiometric decrease in diadinoxanthin. The UV-B dependent increase in diatoxanthin is correlated with a concommitant enhancement of non-photochemical quenching of chlorophyll fluorescence and a decrease in the quantum efficiency of oxygen evolution. This indicates that UV-B induced diatoxanthin functions in thermal energy dissipation. Possible scenarios for a stimulation of the diadinoxanthin cycle by UV-B are discussed.     

Recurrent V1–V2 interaction in early visual boundary processing

A majority of cortical areas are connected via feedforward and feedback fiber projections. In feedforward pathways we mainly observe stages of feature detection and integration. The computational role of the descending pathways at different stages of processing remains mainly unknown. Based on empirical findings we suggest that the top-down feedback pathways subserve a context-dependent gain control mechanism. We propose a new computational model for recurrent contour processing in which normalized activities of orientation selective contrast cells are fed forward to the next processing stage. There, the arrangement of input activation is matched against local patterns of contour shape. The resulting activities are subsequently fed back to the previous stage to locally enhance those initial measurements that are consistent with the top-down generated responses. In all, we suggest a computational theory for recurrent processing in the visual cortex in which the significance of local measurements is evaluated on the basis of a broader visual context that is represented in terms of contour code patterns. The model serves as a framework to link physiological with perceptual data gathered in psychophysical experiments. It handles a variety of perceptual phenomena, such as the local grouping of fragmented shape outline, texture surround and density effects, and the interpolation of illusory contours. Received: 28 October 1998 / Accepted in revised form: 19 March 1999     

M protein, a classical bacterial virulence determinant, forms complexes with fibrinogen that induce vascular leakage

Increased vascular permeability is a key feature of inflammatory conditions. In severe infections, leakage of plasma from the vasculature induces a life-threatening hypotension. Streptococcus pyogenes, a major human bacterial pathogen, causes a toxic shock syndrome (STSS) characterized by excessive plasma leakage and multi-organ failure. Here we find that M protein, released from the streptococcal surface, forms complexes with fibrinogen, which by binding to beta2 integrins of neutrophils, activate these cells. As a result, neutrophils release heparin binding protein, an inflammatory mediator inducing vascular leakage. In mice, injection of M protein or subcutaneous infection with S. pyogenes causes severe pulmonary damage characterized by leakage of plasma and blood cells. These lesions were prevented by treatment with a beta2 integrin antagonist. In addition, M protein/fibrinogen complexes were identified in tissue biopsies from a patient with necrotizing fasciitis and STSS, further underlining the pathogenic significance of such complexes in severe streptococcal infections.     

K<Superscript>+</Superscript> transport in the caterpillar intestine epithelium: role of osmolytes for the K<Superscript>+</Superscript>-secretory capacity of the tobacco hornworm midgut

The midgut of the tobacco hornworm, Manduca sexta, actively secretes potassium ions. This can be measured as short-circuit current (I_sc) with the midgut mounted in an Ussing chamber and superfused with a high-K⁺ saline containing as its major osmolyte 166 mM sucrose. Iso-osmotic substitution of sucrose by non-metabolisable compounds (mannitol, urea, NaCl and the polyethylene glycols 200, 400 and 600) led to a dramatic, though reversible, drop in the current. Acarbose, a specific inhibitor of invertase (sucrase) in vertebrates and insects, had no detectable influence on I_sc. Unexpectedly, after replacing sucrose iso-osmotically with the saccharides glucose, fructose, trehalose or raffinose, the K⁺ current could no longer be supported. However, all osmolytes smaller than sucrose (except for NaCl), metabolisable or not, initiated an immediate, quite uniform but transient, increase in I_sc by about 20%, before its eventual decline far below the control value. Hypo-osmotic treatment by omission of sucrose also transiently increased the K⁺ current. Small osmolytes substituted for sucrose caused no transient I_sc stimulation when the epithelium had been challenged before with hypo-osmolarity; however, the eventual decline in I_sc could not be prevented. Our data seem inconsistent with a role of sucrose as energiser or simple osmolyte. Rather, we discuss here its possible role as analogous to that of sucrose in lower eukaryotes or plants, as an extra- and/or intracellular compatible osmolyte that stabilises structure and/or function of the proteins implicated in K⁺ transport.Communicated by G. Heldmaier     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

Mal de Meleda (MDM) caused by mutations in the gene for SLURP-1 in patients from Germany,Turkey, Palestine,and the United Arab Emirates

Mal de Meleda (MDM) or keratosis palmoplantaris transgrediens of Siemens is an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis with an onset in early infancy. There is no associated involvement of other organs; however, a spectrum of clinical presentations with optional and variable features has been described. Mutations in the ARS (component B)-81/s gene ( LY6LS) on chromosome 8q24-qter, which encodes SLURP-1, have recently been identified in patients with MDM. Here, we have analyzed four MDM families for mutations in SLURP-1. In a large Palestinian pedigree with multiple consanguinity, patients are homozygous for a new mutation that substitutes an arginine for a conserved glycine residue at position 86. A different mutation in Turkish patients results in the same amino acid exchange. Some remarkable similarities are seen in the clinical picture of patients from both families. Patients of an Emirati Bedouin family have a homozygous alteration of the translation initiation codon. In a German family with no known consanguinity, we have shown pseudodominant inheritance. Three affected children and their affected mother are homozygous for the missense mutation W15R. Our findings indicate that the MDM type of transgressive PPK is caused by SLURP-1 mutations in patients from various origins and demonstrate allelic heterogeneity for mutations in SLURP-1.     

Real-time single cell analysis of Smac/DIABLO release during apoptosis

We examined the temporal and causal relationship between Smac/DIABLO release, cytochrome c (cyt-c) release, and caspase activation at the single cell level during apoptosis. Cells treated with the broad-spectrum caspase inhibitor z-VAD-fmk, caspase-3 (Casp-3)-deficient MCF-7 cells, as well as Bax-deficient DU-145 cells released Smac/DIABLO and cyt-c in response to proapoptotic agents. Real-time confocal imaging of MCF-7 cells stably expressing Smac/DIABLO-yellow fluorescent protein (YFP) revealed that the average duration of Smac/DIABLO-YFP release was greater than that of cyt-c-green fluorescent protein (GFP). However, there was no significant difference in the time to the onset of release, and both cyt-c-GFP and Smac/DIABLO-YFP release coincided with mitochondrial membrane potential depolarization. We also observed no significant differences in the Smac/DIABLO-YFP release kinetics when z-VAD-fmk-sensitive caspases were inhibited or Casp-3 was reintroduced. Simultaneous measurement of DEVDase activation and Smac/DIABLO-YFP release demonstrated that DEVDase activation occurred within 10 min of release, even in the absence of Casp-3.     

Sinorhizobium meliloti acpXL mutant lacks the C28 hydroxylated fatty acid moiety of lipid A and does not express a slow migrating form of lipopolysaccharide

Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. Lipid A of all Rhizobiaceae is acylated with a long fatty acid chain, 27-hydroxyoctacosanoic acid. Biosynthesis of this long acyl substitution requires a special acyl carrier protein, AcpXL, which serves as a donor of C28 (omega-1)-hydroxylated fatty acid for acylation of rhizobial lipid A (Brozek, K.A., Carlson, R.W., and Raetz, C. R. (1996) J. Biol. Chem. 271, 32126-32136). To determine the biological function of the C28 acylation of lipid A, we constructed an acpXL mutant of Sinorhizobium meliloti strain 1021. Gas-liquid chromatography and mass spectrometry analysis of the fatty acid composition showed that the acpXL mutation indeed blocked C28 acylation of lipid A. SDS-PAGE analysis of acpXL mutant LPS revealed only a fast migrating band, rough LPS, whereas the parental strain 1021 manifested both rough and smooth LPS. Regardless of this, the LPS of parental and mutant strains had a similar sugar composition and exposed the same antigenic epitopes, implying that different electrophoretic profiles might account for different aggregation properties of LPS molecules with and without a long acyl chain. The acpXL mutant of strain 1021 displayed sensitivity to deoxycholate, delayed nodulation of Medicago sativa, and a reduced competitive ability. However, nodules elicited by this mutant on roots of M. sativa and Medicago truncatula had a normal morphology and fixed nitrogen. Thus, the C28 fatty acid moiety of lipid A is not crucial, but it is beneficial for establishing an effective symbiosis with host plants. acpXL lies upstream from a cluster of five genes, including msbB (lpxXL), which might be also involved in biosynthesis and transfer of the C28 fatty acid to the lipid A precursor.     

Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity,cell survival,and mitochondria number in CHO-DG44 grown in suspension and serum-free media

We have engineered dihydrofolate reductase-negative (dhfr-/-) Chinese hamster ovary (CHO) DG44 cells adapted for growth in serum-free suspension cultures for simultaneous expression of the common cold therapeutic, the soluble intercellular adhesion molecule 1 (sICAM), and the antiapoptosis determinants bcl-2 or bcl-x(L). Detailed analyses of titer and antiapoptosis characteristics of these production cell lines included an independent (sICAM; bcl-2/bcl-x(L)) as well as a cocistronic (sICAM-(bcl-2/bcl-x(L))) expression set-up in which translation-initiation of the survival cistron is driven by an internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). In transient transfections or stable mixed populations and in comparison to isogenic sICAM-only control vectors, both bcl-x(L)-encoding configurations achieved higher sICAM yields while bcl-2 over-expression resulted in decreased product levels. Overall, the death-protective impact of bcl-2 and bcl-x(L) in engineered CHO-DG44 was not significant under typical batch-mode operation, an observation that was confirmed by clonal analysis. bcl-2 and bcl-x(L) displayed their antiapoptosis potential only following dhfr-based amplification in sICAM-producing CHO-DG44 cell lines. In all cases, bcl-x(L) outperformed bcl-2 in its cell death-protective capacity. Amplification-dependent high-level expression of mitochondria-localized bcl-2 family members required for successful antiapoptosis engineering may be essential to compensate for increased mitochondria numbers found to be associated with production cell lines grown in serum-free medium.     

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.