Identification of the abiotic stress-related transcription in little Neptune grass Cymodocea nodosa with RNA-seq.

Seagrasses exhibit vital ecological roles in the marine environment. Nevertheless, the genomic resources available for seagrasses are still scarce. In the present study, the transcriptome of Cymodocea nodosa was sequenced with a view to study the molecular mechanisms underlying abiotic stress responses. The sequenced transcriptome for the species was near-complete and a high percentage of the transcripts was computationally annotated. An experimental simulation of marine plant exposure to extreme temperature (34°C), salinity (50psu) and their combination was conducted. A dynamic transcriptome 24h response (short-term) from stress initialization was recorded. The most noteworthy alteration in gene expression was observed in heat-stressed plants. Transcripts associated with development, photosynthesis, osmotic balance and stress-response were differentially expressed, under the set experimental conditions. Results indicate a potential negative interaction of heat and osmotic stress on seagrasses transcriptome.

Cytoskeleton organisation during the infection of three brown algal species, Ectocarpus siliculosus, Ectocarpus crouaniorum and Pylaiella littoralis, by the intracellular marine oomycete Eurychasma dicksonii.

Oomycete diseases in seaweeds are probably widespread and of significant ecological and economic impact, but overall still poorly understood. This study investigates the organisation of the cytoskeleton during infection of three brown algal species, Pylaiella littoralis, Ectocarpus siliculosus, and Ectocarpus crouaniorum, by the basal marine oomycete Eurychasma dicksonii. Immunofluorescence staining of tubulin revealed how the development of this intracellular biotrophic pathogen impacts on microtubule (MT) organisation of its algal host. The host MT cytoskeleton remains normal and organised by the centrosome until very late stages of the infection. Additionally, the organisation of the parasite's cytoskeleton was examined. During mitosis of the E. dicksonii nucleus the MT focal point (microtubule organisation centre, MTOC, putative centrosome) duplicates and each daughter MTOC migrates to opposite poles of the nucleus. This similarity in MT organisation between the host and pathogen reflects the relatively close phylogenetic relationship between oomycetes and brown algae. Moreover, actin labelling with rhodamine-phalloidin in E. dicksonii revealed typical images of actin dots connected by fine actin filament bundles in the cortical cytoplasm. The functional and phylogenetic implications of our observations are discussed.

Multi-exon deletions of the PKHD1 gene cause autosomal recessive polycystic kidney disease (ARPKD).

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the PKHD1 (polycystic kidney and hepatic disease 1) gene on chromosome 6p12, a large gene spanning 470 kb of genomic DNA. So far, only micromutations in the 66 exons encoding the longest open reading frame (ORF) have been described, and account for about 80% of mutations. OBJECTIVE: To test the hypothesis that gross genomic rearrangements and mutations in alternatively spliced exons contribute to a subset of the remaining disease alleles. METHODS: Using DHPLC for alternatively spliced exons and quantitative real time polymerase chain reaction to detect genomic imbalances, 58 ARPKD patients were screened, of whom 55 were known to harbour one PKHD1 point mutation in the longest ORF. RESULTS: Three different heterozygous PKHD1 deletions and several single nucleotide changes in alternatively spliced exons were identified. The detected partial gene deletions are most likely pathogenic, while a potential biological function of the alterations identified in alternatively spliced exons must await the definition of transcripts containing alternative exons and their predicted reading frames. CONCLUSIONS: Gross PKHD1 deletions account for a detectable proportion of ARPKD cases. Screening for major genomic PKHD1 rearrangements will further improve mutation analysis in ARPKD.

Thrombin-stimulated glutamate uptake in human platelets is predominantly mediated by the glial glutamate transporter EAAT2.

Glutamate toxicity has been implicated in the pathogenesis of various neurological diseases. Glial glutamate transporters play a key role in the regulation of extracellular glutamate levels in the brain by removing glutamate from the extracellular fluid. Since human blood platelets possess an active glutamate uptake system, they have been used as a peripheral model of glutamate transport in the central nervous system (CNS). The present study is aimed at identifying the glutamate transporter on blood platelets, and to asses the influence of platelet activation on glutamate uptake. Platelets from healthy donors showed Na+-dependent glutamate uptake (Km, 3.5+/-0.9 microM; Vmax, 2.8+/-0.2 pmol glutamate/75 x 10(6)platelets/30 min), which could be blocked dose-dependently by the EAAT specific inhibitors DL-threo-E-benzyloxyaspartate (TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and high concentrations of the EAAT2 inhibitor dihydrokainate (DHK). Analysis of platelet homogenates on Western blots showed EAAT2 as the predominant glutamate transporter. Platelet activation by thrombin caused an increase in glutamate uptake, which could be inhibited by TBOA and the EAAT2 inhibitor DHK. Kinetic analysis showed recruitment of new transporters to the membrane. Indeed, Western blot analysis of subcellular fractions revealed that alpha-granules, which fuse with the membrane upon thrombin stimulation, contained significant EAAT2 immunoreactivity. Inhibition of the second messengers involved in alpha-granule secretion (protein kinase C, phosphatidylinositol-3-kinase) inhibited thrombin-stimulated uptake, but not basal uptake. These data show that the glial EAAT2 is the predominant glutamate transporter on blood platelets and suggest, that thrombin increases glutamate uptake capacity by recruiting new transporters (EAAT2) from alpha-granules.

Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata.

Oligomeric degradation products of alginate elicited a respiratory and oxidative burst in the sporophytes of the kelp Laminaria digitata. The generation of activated oxygen species (AOS), O(2)(-), and H(2)O(2) was detected at the single cell level, using nitroblue tetrazolium precipitation and a redox-sensitive fluorescent probe, respectively. The oxidative burst involved diphenyleneiodonium-sensitive AOS-generating machinery and its amplitude depended on the type of tissue. After a first elicitation plants were desensitized for about 3 h. The activity of alginate oligosaccharides was dose dependent, saturating around 40 microM. It was also structure-dependent, with homopolymeric blocks of alpha-1,4-L-guluronic acid, i.e. the functional analogs of oligogalacturonic blocks in pectins, being the most active signals. The perception of oligoguluronate signals resulted in a strong efflux of potassium. Pharmacological dissection of the early events preceding the emission of AOS indicated that the transduction chain of oligoguluronate signals in L. digitata is likely to feature protein kinases, phospholipase A(2), as well as K(+), Ca(2+), and anion channels.

Photometric method for the quantification of chlorophylls and their derivatives in complex mixtures: fitting with Gauss-peak spectra.

Accurate quantification of pigments in mixtures is essential in all cases in which separation of pigments by chromatography is impracticable for one reason or another. An example is the analysis of in vivo formation of heavy metal-substituted chlorophylls in heavy metal-stressed plants. We describe here a novel, accurate UV/VIS spectrophotometric method for the quantification of individual chlorophyll derivatives in complex mixtures, which has the potential for universal applicability for mixtures difficult to separate. The method is based on the description of each pigment spectrum by a series of Gaussian peaks. A sample spectrum is then fitted by a linear combination of these "Gauss-peak spectra" including an automatic correction of wavelength inaccuracy and baseline instability of the spectrometer as well as a correction of the widening of absorbance peaks in more concentrated pigment solutions. The automatic correction of peak shifts can also partially correct shifts caused by processes like allomerization. In this paper, we present the Gauss-peak spectra for Mg-chlorophyll a, b, c, pheophytin a, b, c, Cu-chlorophyll a, b, c, and Zn-chlorophyll a in acetone; Mg-chlorophyll a, b, pheophytin a, b, Cu-chlorophyll a, b, allomerized Cu-chlorophyll a, b, and Zn-chlorophyll a, b in cyclohexane; Mg-chlorophyll a, b, pheophytin a, b, and Cu-chlorophyll a, b in diethyl ether.

Oligosaccharide recognition signals and defence reactions in marine plant-microbe interactions.

Recent findings on the involvement of oligosaccharide signals in pathogen recognition and defence reactions in marine algae shine a new light on the ecology of their interactions with associated microorganisms. Since the marine environment encompasses lineages that have diverged a long time ago from the terrestrial phyla, these results suggest that cell-cell recognition pathways typical of terrestrial plants appeared very early in the evolution of eukaryotes. Production of oligosaccharides from marine algae using microbial recombinant polysaccharidases is also of industrial interest as plants can be protected from infections by preincubation in the presence of appropriate signals that mimic the attacks by pathogens.

A phase-II study with idarubicin, ifosfamide and VP-16 (IIVP-16) in patients with refractory or relapsed aggressive and high grade non-Hodgkin's lymphoma.

We report a phase-II study of idarubicin, ifosphamide and etoposide (IIVP-16) in heavily pretreated patients with relapsed or refractory aggressive non-Hodgkin's lymphoma. The IIVP-16 regimen consisted of idarubicin (10 mg/m2 i.v. days 1 + 2 or days 1 + 8), ifosfamide (1000 mg/m2 i.v. days 1-5) and VP-16 (150 mg/m2 i.v. days 103). 40 patients were enrolled. Of 38 evaluable patients, 26 had centroblastic subtype, followed by lymphoblastic (6), immunoblastic (2), and other entities (4). 18 patients were primary resistant; 14 patients had early relapse (CR less than 12 months) and 8 patients late relapse (CR longer than 12 months). The median number of different prior chemotherapy regimens was 2 (range 1 to 6). 20 patients had received additional radiotherapy. The response-rate was 47.4% including 8 CR (21.1%) and 10 PR (26.3%). IIVP-16 was more effective in patients with relapsed disease when compared with patients with primary resistant disease (response rate 65% vs. 27.8%, p < 0.025). Leukopenia and thrombocytopenia were the major toxicities occurring in 73/107 (68.2%) and 57/107 (53.3%) of cycles (WHO grade IV). IIVP-16 is an effective regimen particularly in patients with unfavorable relapsed NHL.

[Effect of etomidate and thiopental on ACTH and cortisol levels in serum. A prospective controlled comparative study in healthy probands]. / Der Einfluss von Etomidat und Thiopental auf ACTH- und Cortisolspiegel im Serum. Eine prospektive kontrollierte Vergleichsuntersuchung an gesunden Probanden.

A prospective controlled trial was carried out to determine the influence of etomidate and thiopentone on serum levels of cortisol and plasma levels of adrenocorticotrophic hormone (ACTH). There were two groups of nine healthy male volunteers: one group was given 0.3 mg/kg body wt. etomidate and the other 4 mg/kg body wt. thiopentone intravenously as a bolus. The hormone levels were measured before and every 30 min after injection of the anaesthetic over a period of 5 h. After thiopentone, cortisol levels dropped from a mean value of 13.2 micrograms/dl to a minimum of 9.0 micrograms/dl after 90 min and fluctuated between 9.2 and 10.9 micrograms/dl until the end of the experiment. Etomidate induced a more pronounced decrease in cortisol levels (from the 90th min the differences to the levels after thiopentone were statistically significant) from 13.9 micrograms/dl to a minimum of 5.0 micrograms/dl after 150 min. After 300 min cortisol levels had recovered only to 7.7 micrograms/dl. Plasma levels of ACTH were also different in the two groups. After thiopentone they decreased from 26.1 pg/ml to 18.2 pg/ml after 30 min, then increased to 30 pg/ml after 300 min. After etomidate, ACTH levels were as high at 30 min as before the injection and increased continuously to 67.1 pg/ml by the end of the experiment; from the 150th min on, the differences to the ACTH levels after thiopentone were statistically significant. We conclude from these hormone levels that a single intravenous bolus injection of thiopentone induces a slight but statistically significant and ACTH-independent decrease in adrenal cortisol secretion in healthy volunteers not stressed by an operation.(ABSTRACT TRUNCATED AT 250 WORDS)