Chlorophyll a Fluorescence Predicts Total Photosynthetic Electron Flow to CO(2) or NO(3)/NO(2) under Transient Conditions

A model which predicts total photosynthetic electron flow from a linear regression of the relationship between corrected steady-state quantum yield and nonphotochemical quenching (E Weis, JA Berry [1987] Biochem Biophys Acta 894: 198-208) was formulated for N-limited cells of the green alga Selenastrum minutum. Unlike other models based on net CO₂ fixation, our model is based on total photosynthetic electron flow measured as gross O₂ evolution. This allowed for the prediction of total photosynthetic electron flow from water to both CO₂ fixation and NO₃⁻/NO₂⁻ reduction. The linear regression equation predicting electron flow is of the form: J = I · Q_q[0.4777-0.3282 Q_NP] (where J = gross photosynthetic electron flow, I = incident PAR, Q_q = photochemical quenching, Q_NP = nonphotochemical quenching). During steady-state photosynthesis, over a range of irradiance, the model predicted a photosynthetic light saturation curve which was well correlated with that observed. Although developed under steady-state conditions, the model was tested during nonsteady-state photosynthesis induced by transient nitrogen assimilation. The model predicted transient rates of gross O₂ evolution which were in excellent agreement with the rates observed under a variety of conditions regardless of whether CO₂ or NO₃⁻/NO₂⁻ served as the physiological electron acceptor. The fluorescence transients resulting from ammonium and nitrate assimilation are discussed with respect to metabolic demands for reductant and ATP.     

Siderophores and outer membrane proteins of antagonistic, plant-growth-stimulating, root-colonizing Pseudomonas spp

As an approach to understanding the molecular basis of the reduction in plant yield depression by root-colonizing Pseudomonas spp. and especially of the role of the bacterial cell surfaces in this process, we characterized 30 plant-root-colonizing Pseudomonas spp. with respect to siderophore production, antagonistic activity, plasmid content, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of their cell envelope proteins. The results showed that all strains produce hydroxamate-type siderophores which, because of the correlation with Fe3+ limitation, are thought to be the major factor responsible for antagonistic activity. Siderophore-negative mutants of two strains had a strongly decreased antagonistic activity. Five strains maintained their antagonistic activity under conditions of iron excess. Analysis of cell envelope protein patterns of cells grown in excess Fe3+ showed that most strains differed from each other, although two classes of similar or identical strains were found. In one case such a class was subdivided on the basis of the patterns of proteins derepressed by iron limitation. Small plasmids were not detected in any of the strains, and only one of the four tested strains contained a large plasmid. Therefore, it is unlikely that the Fe3+ uptake system of the antagonistic strains is usually plasmid encoded.     

Cytochrome and alternative pathway respiration in green algae : measurements using inhibitors and o(2) discrimination

Inhibitor titration curves and discrimination against ¹⁸O₂ by mitochondrial respiration in three strains of green algae (Selenastrum minutum [Naeg.] Collins, and two strains of Chlamydomonas reinhardtii Dangeard) with differing respiratory capabilities were determined. Discrimination for cytochrome pathway respiration ranged from 19.89 to 20.43%. Discrimination for alternative pathway respiration by wild-type C. reinhardtii (measured in the presence of KCN) was 25.46%, while discrimination values for a cytochrome oxidase deficient mutant of C. reinhardtii ranged from 24.24 to 24.96%. In the absence of KCN, the alternative pathway was not engaged in wild-type C. reinhardtii, the only algal strain that possessed both cytochrome and alternative pathway capacities.     

An alternatively spliced Interleukin 4 form in lymphoid cells

The nucleotide sequence data presented in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X81851     

Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs

Theodorou, Andreas, Natalie Weger, Kathleen Kunke, KyooRhee, David Bice, Bruce Muggenberg, and Richard Lemen. Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs. J. Appl. Physiol.83(3): 912-917, 1997.In ragweed (RW)-sensitized beagle dogs, wetested the hypothesis that reactivity of the pulmonary vasculature wasenhanced with aerosolized histamine (Hist) and RW. Seven dogs wereneonatally sensitized with repeated intraperitoneal RW injections, and12 dogs were controls (Con). The dogs were anesthetizedwith intravenous chloralose, mechanically ventilated, and instrumentedwith femoral arterial and pulmonary artery catheters. Specific lungcompliance(CL_sp),specific lung conductance (G_sp),systemic vascular resistance index, and pulmonary vascular resistanceindex (PVRI) were measured before and after bronchoprovocation withHist and RW. After Hist inhalation (5 breaths of 30 mg/ml), both Conand RW dogs had significant (P < 0.05) decreases inCL_sp(51 ± 4 and 53 ± 5%, respectively) andG_sp (65 ± 5 and69 ± 3%, respectively), but only RW-sensitized dogs had asignificant increase in PVRI (38 ± 10%). After RW inhalation (60 breaths of 0.8 mg/ml), only RW-sensitized dogs had significant increases (62 ± 20%) in PVRI and decreases inG_sp (77 ± 4%) and CL_sp(65 ± 7%). We conclude that, compared with Con,RW-sensitized beagle dogs have increased pulmonary vasoconstrictiveresponses with Hist or RW inhalation.

     

Use of Bioluminescence Markers To Detect Pseudomonas spp. in the Rhizosphere

The use of bioluminescence as a sensitive marker for detection of Pseudomonas spp. in the rhizosphere was investigated. Continuous expression of the luxCDABE genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible luxCDABE construct or a constitutive luxAB construct (coding only for the luciferase) was introduced into the Pseudomonas cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase, n-decyl aldehyde, respectively. These Pseudomonas cells could successfully be detected in the rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 10³ to 10⁴ CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than β-galactosidase-based systems.     

Enzyme-cytochemistry of the saccus dorsalis of the rainbow trout,Salmo gairdneri Richardson

Summary In the saccus dorsalis of the rainbow trout, Salmo gairdneri Richardson, the activity of various enzymes (transferase, lyases, oxidoreductases, hydrolases) have been studied in detail.The results of this enzyme-cytochemical study firmly demonstrate that the organ is metabolically highly active. The epithelial cells have a strong energy metabolism. Energy production can take place under aerobic as well as under anaerobic conditions. Evidence is presented that glucose from blood is directly utilized for energy demands. The epithelial cells show also high synthetic activities. The moderate amino acid metabolism may participate in the synthesis of an acid mucopolysaccharide-protein complex, especially in the so-called dark cells. Lipid metabolism appears to be restricted to the mitochondria, indicating a high turnover of lipid moieties in the membranes. In contrast to the normal looking mitochondria, the macromitochondria — besides shape and localization — have an extremely high lipid and monoamine metabolism, which may point to a special function in the cellular economy. The high activity of enzymes involved in the degradation of monoamines and in the hydration of CO₂ is of particular physiological interest. The significance of the observations is discussed in relation to formerly obtained indications on the involvement of the saccus dorsalis in fluid secretion, extrusion of organic substances of low molecular weight into the ventricular system and uptake of organic substances from the cerebrospinal fluid.The hypothesis of the saccus dorsalis being an analogue of the choroid plexus is supported by several relevant data.     

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

OXYGEN CONSUMPTION ASSOCIATED WITH FERRIC REDUCTASE ACTIVITY AND IRON UPTAKE BY IRON-LIMITED CELLS OF CHLORELLA KESSLERI (CHLOROPHYCEAE)

Plasma membrane ferric reductase activity was enhanced 5-fold under iron limitation in the unicellular green alga Chlorella kessleri Fott et Nováková. Furthermore, ferric reductase activity in iron-limited cells was approximately 50% higher in the light than in the dark. In contrast, iron uptake rates of iron-limited cells were unaffected by light versus dark treatments. Rates of iron uptake were much lower than rates of ferric reduction, averaging approximately 2% of the dark ferric reduction rate. Ferric reduction was associated with an increased rate of O₂ consumption in both light and dark, the increase in the light being approximately 1.5 times as large as in the dark. The increased rate of O₂ consumption could be decreased by half by the addition of catalase, indicating that H₂O₂ is the product of the O₂ consumption and that the increased O₂ consumption is nonrespiratory. The stimulation of O₂ consumption was almost completely abolished by the addition of bathophenanthroline disulfonate, a strong chelator of Fe^{2 +} . Anaerobic conditions or the presence of exogenous superoxide dismutase affected neither ferric reduction nor iron uptake. We suggest that the O₂ consumption associated with ferric reductase activity resulted from superoxide formation from the aerobic oxidation of Fe^{2 +} , which is the product of ferric reductase activity. At saturating concentrations of Fe^{3 +} chelates, ferric reductase activity is much greater than the iron uptake rate, leading to rapid oxidation of Fe^{2 +} and superoxide generation. Therefore, O₂ consumption is not an integral part of the iron assimilation process.