Cell physiology and antibiotic production of Streptomyces coelicolor grown on solid medium

Summary In order to examine the physiology ofStreptomyces coelicolor when growing on solid media, we have employed a membrane overlay technique and used a new approach to extract substrate and product compounds from the agar. Comparisons made with liquid grown cultures indicate a change from non-growth associated productivity of actinorhodin in liquid culture, to growth associated production on agar plates. In contrast, the temporal control of methylenomycin production was virtually identical under both culture conditions. Considerable extracellular protein production was observed during growth on agar.     

Simultaneous measurement of changes in red and blue fluorescence in illuminated isolated chloroplasts and leaf pieces: The contribution of NADPH to the blue fluorescence signal

A newly developed nitrogen laser fluorimeter insensitive to actinic illumination was used to follow simultaneously the light induced changes in red and blue fluorescence of intact isolated spinach chloroplasts and leaf pieces. The recorded variable blue fluorescence was linked to a water soluble component of intact isolated chloroplasts, depended on Photosystem I, and was related to changes in carbon metabolism. From the comparison of changes in intact and broken chloroplasts and from fluorescence spectra under different conditions, it was concluded that the variation in NADPH was the major cause for the changes in blue fluorescence. This study opens a path towards continuous and non-destructive monitoring of NADPH redox state in chloroplasts and leaves.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - DLGA DL-glyceraldehyde - FNR ferredoxin-NADP reductase - FWHM full width at half maximum - LED light emitting diodes - OAA oxaloacetate - q_N non-photochemical quenching - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - q_P photochemical quenching - PPFD photosynthetic photon flux density - Q_A primary quinone acceptor of Photosystem II Preliminary results of this work were presented at the First Conference on the Physiology and Biochemistry of high Mountain Plants, 2–3 July 1992, Villar d'Arene, France.     

Differential expression of several E2-type ubiquitin carrier protein genes at different developmental stages inArabidopsis thaliana andNicotiana sylvestris

We characterized three genes encoding different E2-type ubiquitin carrier proteins involved in the ubiquitin-mediated proteolytic pathway:UbcAt3 shows homologies to the yeastCDC34 gene andUbcAt4a andUbcAt4b are two different genes homologous to theUbc1/4/5 subfamily in yeast. Their accumulation was analysed and compared with that of the different families encoding polyubiquitins, as well as the monoubiquitin fusion protein, which is considered as a marker for cell division, during various developmental stages including GO/S transition and senescence of higher plant cells. Our results imply that theseUbc genes are under the control of complex mechanisms, and are differentially regulated, but not necessarily co-regulated with ubiquitin genes. Even the closely relatedUbcAt4a andUbcAt4b genes of the same multigene subfamily are controlled by distinct regulatory mechanisms.     

Submerged vascular plants and water chemistry in the coastal marsh Albufera de Mallorca (Balearic Islands)

Ten species of aquatic macrophytes have been analyzed for seven environmental variables by means of a significance test. A synthetic view has been obtained through a cluster analysis for species and a principal component analysis for 17 variables which form the multidimensional space where we project the species habitats. Myriophyllum spicatum L., Najas marina L., Potamogeton crispus L., Potamogeton pectinatus L. and Zannichellia pedunculata Reichenb. are widely distributed in the Albufera. Ceratophyllum submersum L. and Ruppia maritima L. var. brevirostris Ag. are considered stenoic. The tolerance of Ceratophyllum submersum to salts is significantly low and that of Ruppia cirrhosa (Petagna) Grande and Ruppia maritima var. brevirostris significantly high. Ceratophyllum submersum has a significantly negative distribution with regard to chlorophyll a and phosphate concentrations. Ceratophyllum demersum L. and C. submersum primarily occur in nitrate-rich waters whereas Ruppia cirrhosa primarily occurs in low nitrate waters.     

Genetic bottlenecks and population passages cause profound fitness differences in RNA viruses.

Repeated clone-to-clone (genetic bottleneck) passages of an RNA phage and vesicular stomatitis virus have been shown previously to result in loss of fitness due to Muller's ratchet. We now demonstrate that Muller's ratchet also operates when genetic bottleneck passages are carried out at 37 rather than 32 degrees C. Thus, these fitness losses do not depend on growth of temperature-sensitive (ts) mutants at lowered temperatures. We also demonstrate that during repeated genetic bottleneck passages, accumulation of deleterious mutations does occur in a stepwise (ratchet-like) manner as originally proposed by Muller. One selected clone which had undergone significant loss of fitness after only 20 genetic bottleneck passages was passaged again in clone-to-clone series. Additional large losses of fitness were observed in five of nine independent bottleneck series; the relative fitnesses of the other four series remained close to the starting fitness. In sharp contrast, when the same selected clone was transferred 20 more times as large populations (10(5) to 10(6) PFU transferred at each passage), significant increases in fitness were observed in all eight passage series. Finally, we selected several clones which had undergone extreme losses of fitness during 20 bottleneck passages. When these low-fitness clones were passaged many times as large virus populations, they always regained very high relative fitness. We conclude that transfer of large populations of RNA viruses regularly selects those genomes within the quasispecies population which have the highest relative fitness, whereas bottleneck transfers have a high probability of leading to loss of fitness by random isolation of genomes carrying debilitating mutations. Both phenomena arise from, and underscore, the extreme mutability and variability of RNA viruses.     

Enumeration of Obesumbacterium proteus in brewery yeasts and characterization of isolated strains using Biolog GN microplates and protein fingerprinting

Of a range of media tested for enumeration of Obesumbacterium proteus in brewers' yeast, Universal Beer agar and Wallerstein Laboratories Differential medium were most effective. MacConkey agar (several types) and Membrane Lauryl Sulphate agar were least effective. Other media (Wort agar, YM agar) were of intermediate efficacy. Nine O. proteus strains from commercial yeast samples were characterized using the API 20E test kit, the Biolog GN microplate (BGNM) and by SDS-PAGE of their total soluble proteins. Both the BGNM and SDS-PAGE techniques allowed the strains to be differentiated from one another: the API 20E kit did not. All strains isolated from UK breweries belonged to O. proteus biogroup II. Four of these strains displayed a branching cell morphology not hitherto described in any member of the Enterobacteriaceae.     

Immunomagnetic separation as a final purification step of liver endothelial cells

Summary We describe a fast and reproducible method that can be used as a final step in obtaining pure populations of liver endothelial cells. This method employs endothelial cell specific lectin covalently bound to magnetic polystyrene beads (Dynabeads). Evonymus europaeus agglutinin (EEA)-coated Dynabeads were used to purify monkey liver endothelium from Percoll gradient separated nonparenchymal cells. EEA-coated beads were also successfully used to purify monkey aortic endothelial cells. The endothelial cells grew to confluence as a cobblestonelike monolayer, expressed Factor VIII related antigen, and incorporated acetylated-low density lipoprotein. The magnetic beads seemed not to modify the normal properties of the isolated endothelium, thus facilitating their use in experimental studies. This immunomagnetic separation technique may be applicable for purification of endothelial cells from a wide variety of tissue sources.     

Oral nicotinamide riboside raises NAD+ and lowers biomarkers of neurodegenerative pathology in plasma extracellular vesicles enriched for neuronal origin

Declining nicotinamide adenine dinucleotide (NAD⁺) concentration in the brain during aging contributes to metabolic and cellular dysfunction and is implicated in the pathogenesis of aging-associated neurological disorders. Experimental therapies aimed at boosting brain NAD⁺ levels normalize several neurodegenerative phenotypes in animal models, motivating their clinical translation. Dietary intake of NAD⁺ precursors, such as nicotinamide riboside (NR), is a safe and effective avenue for augmenting NAD⁺ levels in peripheral tissues in humans, yet evidence supporting their ability to raise NAD⁺ levels in the brain or engage neurodegenerative disease pathways is lacking. Here, we studied biomarkers in plasma extracellular vesicles enriched for neuronal origin (NEVs) from 22 healthy older adults who participated in a randomized, placebo-controlled crossover trial (NCT02921659) of oral NR supplementation (500 mg, 2x /day, 6 weeks). We demonstrate that oral NR supplementation increases NAD⁺ levels in NEVs and decreases NEV levels of Aβ42, pJNK, and pERK1/2 (kinases involved in insulin resistance and neuroinflammatory pathways). In addition, changes in NAD(H) correlated with changes in canonical insulin–Akt signaling proteins and changes in pERK1/2 and pJNK. These findings support the ability of orally administered NR to augment neuronal NAD⁺ levels and modify biomarkers related to neurodegenerative pathology in humans. Furthermore, NEVs offer a new blood-based window into monitoring the physiologic response of NR in the brain.