Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from aKlebsiella pneumoniae mutant strain

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA⁺ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.     

Effect of gibberellins and the growth retardant CCC on the nodulation of soya

Summary The effect of exogenous applications of gibberellins (GAs) or the growth retardant -chloroethyltrimethylammonium chloride (CCC) on root nodule formation and activity (C₂H₂-reduction) in soya was studied. Daily foliar application of GA₃ (2.89×10^–6 M) delayed the formation of nodule initials and reduced the numbers mass nodule^–1 and specific activity of nodules by 43%, 31% and 47% respectively, without affecting plant growth. Similar effects on nodulation were produced by foliar application of GA₄ (3.01×10^–5 M) or GA₇ (3.03×10^–5 M), or by the addition of GA₃ (2.89×10^–6 M) to the rooting medium. GA effectiveness in reducing nodule numbers was decreased by delaying its application until after the initial infection process had occurred, but the nodules formed were smaller and less active than those of the untreated control plants. The GA effect on nodulation and nodule activity was not associated with alterations in root exudate or due to a direct inhibitory effect of the hormone on the nitrogenase system. When the endogenous root content of GA-like substances was reduced (86% decrease) by foliar application of CCC (6.30×10^–5 M), nodule numbers were increased by 56%, but nodule size and total nodule activity were similar to those of control plants. The GA and CCC treatments had no effect on rhizobial growth in liquid culture nor on root colonisation by rhizobia.The results suggest that the endogenous content of root GA may have a regulatory role in both the infection process and in subsequent nodule morphogenesis, thus controlling both the number and effectiveness of the root nodules formed.     

Abscisic acid and gibberellin-like substances in roots and root nodules ofGlycine max

Summary The content of endogenous gibberellin (GA)-like substances of roots and root nodules of SOya, and GA production byRhizobium japonicum cultures, were investigated by a combined thin layer chromatographic (TLC)-dwarf pea epicotyl bioassay technique. GAs were more concentrated in root nodules than in the roots, totalling 1.34 and 0.16 nM GA₃ equivalents g⁻¹ dry wt. respectively. GA production byR. japonicum cultures was demonstrated (1.00 nM GA₃ equivalentsl ⁻¹) and comparison of the GA components of plant and bacterial culture medium extracts, suggested that rhizobial GA production may contribute to the nodule GA content. Cis-trans abscisic acid (ABA) was identified in root and nodule extracts by TLC-gas liquid chromatography (GLC), and amounted to 0.18 and 2.21 nM g⁻¹ dry wt. respectively, whereas 0.30 and 4.63 nM ABA equivalents g⁻¹ dry wt. were detected by a TLC-wheat embryo bioassay technique. ABA was not detected in extracts of bacterial cultures.     

Electrophoretic transport of Tl+ in mitochondria

Summary The distribution of Tl⁺ between rat liver mitochondria and the medium was studied; millimolar or smaller concentrations of Tl⁺ were labeled with²⁰⁴Tl. The Tl⁺ distribution responded to transient diffusion potentials in a way that indicated electrophoretic movements of Tl⁺. The diffusion potentials were induced by efflux of K⁺ in response to addition of valinomycin to nonrespiring mitochondria suspended in a medium with low concentrations of K⁺ or by efflux of H⁺ induced by making the medium more alkaline in the presence of a protonophorous (proton-conducting) uncoupling agent. Changes in membrane potential induced by valinomycin were followed with the aid of safranine. Tl⁺ brought about collapse of the diffusion potential. It is concluded that Tl⁺ is able to penetrate the mitochondrial membrane electrophoretically.     

Monoamines and Their Precursors and Metabolites in the Chicken Brain, Pineal, and Retina: Regional Distribution and Day/Night Variations

Levels of norepinephrine, epinephrine, dopamine, and serotonin (5-HT) and their precursors [tyrosine, L-3,4-dihydroxyphenylalanine, tryptophan, and 5-hydroxytryptophan (5-HTP)] and metabolites [3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid (5-HIAA)] were determined concurrently in samples of chick retina, pineal gland, and nine selected areas of the brain (optic lobes, thalamus, hypothalamus, optic chiasm, pons/medulla, cerebellum, neostriatum/ectostriatum, hyperstriatum, and basal forebrain) using HPLC coupled with a coulometric electrode array detection system. The norepinephrine level was highest in the pineal gland, but it was also widely distributed throughout the chick brain, with the thalamus and hypothalamus showing substantial levels. The dopamine level was highest in the basal forebrain. The epinephrine level was highest in the hypothalamus. The thalamus and hypothalamus showed the highest levels of 5-HT. Daytime levels (1100 h) of these compounds were compared with levels in chicks killed in the middle of the dark phase (2300 h). In the brain areas examined, no day/night variations in levels of norepinephrine, epinephrine, dopamine, or 5-HT were seen, although significant nocturnal changes in levels of their metabolites were observed in some areas. Pineal levels of 5-HIAA decreased significantly at night. The retina showed significant nocturnal increases in 5-HTP, 5-HT, and 5-HIAA levels. Retinal levels of 3-MT and DOPAC were significantly decreased at night.     

Waterborne transmission ofCampylobacter enteritis

Campylobacter jejuni is an important cause of human diarrheal disease throughout the world and likeSalmonella enteritidis, has a large animal reservoir which includes most of man's domestic animals. Until recently, it has been difficult to trace the chain of transmission from animals to man because of inadequate environmental sampling techniques and means to distinguish strains. Recent improvements in these techniques have made environmental studies more feasible in 2 water-related out-breaks.In 1 study,C. jejuni was found to be an important cause of sporadic, summertime diarrheal disease among hikers in national wilderness areas of Wyoming. In this setting, illness was significantly associated with drinking untreated surface water. SubsequentlyC. jejuni was isolated from surface water, including mountian streams, and from animals in the area. Some of the environmental isolates were serotypically identical to strains isolated from humans.A second study occurred as a result of an outbreak of Campylobacter enteritis in a community in northern Illinois which was epidemiologically associated with the community water system.Campylobacter jejuni was isolated from several surface water sources and from the implicated water system. These studies demonstrate that environmental isolation ofC. jejuni is now possible and may add to our understanding of disease transmission.     

Bio-catalyzed coloration of cellulose fibers

Cotton fabrics were dyed with dyes generated in situ by laccase-catalyzed oxidative coupling of the colorless 2,5-diaminobenzenesulfonic acid (2,5-DABSA) and 1-hydroxyphenol (catechol). The enzymatic oxidation of the dye intermediates led to cross-coupling reaction products when the reaction was conducted with an excess of catechol. At least fourfold excess of catechol was necessary to achieve satisfactory dye fixation on cotton. Formation of the same colored product using either an equimolar ratio of the reagents or tenfold excess of catechol was observed. Most probably, homo-molecular reactions predominate over the cross-coupling at equimolar ratio of the precursors, while with an excess of catechol, the cross-coupling occurs in higher yield. The reaction was followed using UV-Vis spectroscopy, HPLC, FTIR and MALDI-TOF MS. A reaction pathway for laccase-induced cross-coupling of catechol and 2,5-DABSA yielding a major colored product was proposed.     

Ultrastructural changes in naturally aged Pinus pinea seeds

The decline of germination observed after storage of Pinus pinea L. seeds up to 35 years at room temperature was accompanied by an increase in their ability to leak reducing sugars, probably as a consequence of membrane alterations due to senescence. Results of studies of the cell ultrastructure of viable and non-viable seeds before and after imbibition indicate that such is the case. Noticeable changes occurred in the ultrastructure of endosperm and embryo cells with germinability loss, even in the unimbibed condition. The main alterations were spherosome coalescence, plasmalemma separation from the cell wall and chromatin aggregation.     

Gadolinium-containing carbon nanomaterials for magnetic resonance imaging: Trends and challenges

Gadolinium-containing carbon nanomaterials are a new class of contrast agent for magnetic resonance imaging. They are characterized by a superior proton relaxivity to any current commercial gadolinium contrast agent and offer the possibility to design multifunctional contrasts. Intense efforts have been made to develop these nanomaterials because of their potential for better results than the available gadolinium contrast agents. The aim of the present work is to provide a review of the advances in research on gadolinium-containing carbon nanomaterials and their advantages over conventional gadolinium contrast agents. Due to their enhanced proton relaxivity, they can provide a reliable imaging contrast for cells, tissues or organs with much smaller doses than currently used in clinical practice, thus leading to reduced toxicity (as shown by cytotoxicity and biodistribution studies). Their active targeting capability allows for improved MRI of molecular or cellular targets, overcoming the limited labelling capability of available contrast agents (restricted to physiological irregularities during pathological conditions). Their potential of multifunctionality encompasses multimodal imaging and the combination of imaging and therapy.     

Holistic assessment of the microbiome dynamics in the substrates used for commercial champignon (Agaricus bisporus) cultivation

Microorganisms strongly influence and are required to generate the selective substrate that provides nutrients and support for fungal growth, and ultimately to induce mushroom fructification under controlled environmental conditions. In this work, the fungal and bacterial microbiota living in the different substrates employed in a commercial crop (compost phase I, II and III, flush 1 and 2, and casing material on day 1, 6 and 8 after compost casing and during flush 1 and 2) have been characterized along the different stages of cultivation by metataxonomic analysis (16S rRNA and ITS2), analysis of phospholipid fatty acid content (PLFAs) and RT-qPCR. Additionally, laccase activity and the content of lignin and complex carbohydrates in compost and casing have been quantified. The bacterial diversity in compost and casing increased throughout the crop cycle boosted by the connection of both substrates. As reflected by the PLFAs, the total living bacterial biomass appears to be negatively correlated with the mycelium of the crop. Agaricus bisporus was the dominant fungal species in colonized substrates, displacing the pre-eminent Ascomycota, accompanied by a sustained increase in laccase activity, which is considered to be a major product of protein synthesis during the mycelial growth of champignon. From phase II onwards, the metabolic machinery of the fungal crop degrades lignin and carbohydrates in compost, while these components are hardly degraded in casing, which reflects the minor role of the casing for nourishing the crop. The techniques employed in this study provide a holistic and detailed characterization of the changing microbial composition in commercial champignon substrates. The knowledge generated will contribute to improve compost formulations (selection of base materials) and accelerate compost production, for instance, through biotechnological interventions in the form of tailored biostimulants and to design environmentally sustainable bio-based casing materials.