Evaluation of the effectiveness factor along immobilized enzyme fixed-bed reactors: Design of a reactor with naringinase covalently immobilized into glycophase-coated porous glass

A design equation is presented for packed-bed reactors containing immobilized enzymes in spherical porous particles with internal diffusion effects and obeying reversible one-intermediate Michaelis-Menten kinetics. The equation is also able to explain irreversible and competitive product inhibition kinetics. It allows the axial substrate profiles to be calculated and the dependence of the effectiveness factor along the reactor length to be continuously evaluated. The design equation was applied to explain the behavior of naringinase immobilized in Glycophase-coated porous glass operating in a packed-bed reactor and hydrolyzing both p-nitrophenyl-alpha-L-rhamnoside and naringin. The theoretically predicted results were found to fit well with experimentally measured values.     

Galanthamine production in “shoot-clump” cultures of Narcissus confusus in liquid-shake medium

Galanthamine (GAL) is increasingly used in the treatment of Alzheimer's disease. We have attempted to develop a method of producing this alkaloid using in vitro cultures of Narcissus confusus plants. The “shoot-clump” culture in liquid medium was shown to be an appropriate method for the micropropagation of this bulbous plant. The complete process included three steps:

1. culture of “twin-scales” starting from the bulbs;
2. culture of the newly formed shoots in a medium for bud proliferation (Murashige Skoog+1 mg l^-1 of 2,4-dichlorophenoxyacetic acid+5 mg l^-1 of benzyladenine), and
3. culture of “shoot-clumps” in a liquid-shake medium. Here we describe the effect of the addition of trans-cinnamic acid, a precursor in the biosynthesis of the Amaryllidaceae alkaloids, on the production of galanthamine and related alkaloids, and also on the growth of the “shoot-clump” culture. The production of galanthamine was found to be inhibited by the addition of the precursor, which promoted the production of the other alkaloid in the same biosynthetic pathway, N-formyl-norgalanthamine. The total production of galanthamine in the control cultures in day-long photoperiod was 2.50 mg per culture, of which 1.97 mg per culture were released into the medium.

     

Soils in warmer and less developed countries have less micronutrients globally

Soil micronutrients are capital for the delivery of ecosystem functioning and food provision worldwide. Yet, despite their importance, the global biogeography and ecological drivers of soil micronutrients remain virtually unknown, limiting our capacity to anticipate abrupt unexpected changes in soil micronutrients in the face of climate change. Here, we analyzed >1300 topsoil samples to examine the global distribution of six metallic micronutrients (Cu, Fe, Mn, Zn, Co and Ni) across all continents, climates and vegetation types. We found that warmer arid and tropical ecosystems, present in the least developed countries, sustain the lowest contents of multiple soil micronutrients. We further provide evidence that temperature increases may potentially result in abrupt and simultaneous reductions in the content of multiple soil micronutrients when a temperature threshold of 12–14°C is crossed, which may be occurring on 3% of the planet over the next century. Altogether, our findings provide fundamental understanding of the global distribution of soil micronutrients, with direct implications for the maintenance of ecosystem functioning, rangeland management and food production in the warmest and poorest regions of the planet.     

Biogeographical Patterns and Phenological Changes in Lapiedra martinezii Lag. Related to Its Alkaloid Diversity

The aim of this work was to investigate the alkaloid patterns of Lapiedra martinezii and their relation to biogeography and phenology focused in a phylogenetic comparison. Plants from 14 populations of L. martinezii, covering almost its entire distribution area, were subjected to morphological, ecological, and phytochemical analysis. Experiments for different alkaloid‐type content are proposed as a new tool for analysis of plant distribution. Several plants were transplanted for weekly observation of their phenological changes, and alkaloids from different plant organs were extracted, listed, and compared. The alkaloid pattern of L. martinezii comprises 49 compounds of homolycorine, lycorine, tazettine, haemantamine, and narciclasine types. The populations located in the north and south margins of the distribution area displayed alkaloid patterns different from those of the central area. Changes in these patterns during their phenological cycle may be related to a better defence for plant reproduction. L. martinezii is an old relict plant, and it has maintained some of the more primitive morphological features and alkaloid profiles of the Mediterranean Amaryllidaceae. The variations in alkaloid content observed could be interpreted in a phylogenetic sense, and those found in their phenological changes, in an adaptive one.     

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology

The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes (¹³C, ¹⁵N, ³⁶S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.     

Use of adjuvant-enhanced formulations to increase bipyridylium-herbicide effectiveness

Paraquat and diquat are two popular, non-selective; bipyridylium herbicides commonly used in citrus orchards and horticultural row crops as the main chemical weed control method. However, since diquat lacks of an effective spectrum against grass weeds, and paraquat mammal toxicity raises strong environmental concerns, both an increase in diquat toxicity against grasses and a reduction in paraquat rates may be desired. Using grass-weed Lolium rigidum and broad leave weed Portulaca oleracea as experimental systems, the effects of six commercial adjuvants (poly-1-p-menthene, mixture of methyl oleate and palmitate, alkylglycol ester, dodecylbenzene ammonium sulphonate, and two paraffinic oils) on paraquat and diquat effectiveness have been studied under laboratory controlled conditions. Dose-response assays showed that adjuvants failed in increasing paraquat efficacy in both broad and grass weeds, yet antagonistic effects being observed in some mixtures such as paraquat + polymentene. However, all adjuvants tested did succeed in increasing significantly diquat effectiveness in P. oleracea and (most important) L. rigidum grass weed. Formulated-diquat ED50 rates were reduced down to 15% (diquat + DBSA) and 30% (diquat + fatty acid ester, diquat + polimentene) of those obtained on non-formulated-diquat trials for P. oleracea and L. rigidum, respectively. Results showed that formulated diquat proved to be a valid alternative to paraquat, and could be used as a more environmentally friendly substitute with comparable effectiveness and herbicide rate.     

The preovulatory rise of ovarian ornithine decarboxylase is required for progesterone secretion by the corpus luteum

Ovarian progesterone secretion during the diestrus stage of the estrous cycle is produced by luteal cells derived from granulosa and thecal cells after the differentiation process that follows ovulation. Our results show that blockade of the preovulatory rise of ovarian ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by treatment with the specific inhibitor alpha-difluoromethylornithine (DFMO) leads to a significant decrease in the ovarian progesterone content and a dramatic fall in the plasma levels of this hormone during the following diestrus. The same inhibition was produced in spite of the fact that both luteinizing and follicle stimulating hormones were given concomitantly with DFMO. On the other hand, the acute rise in the plasma progesterone levels observed after administration of human chorionic gonadotropin to mice at different periods of the estrous cycle was not affected by DFMO administration. Our results indicate that although elevated levels of ODC are not required for acute ovarian steroidogenesis, the preovulatory peak of ovarian ODC activity observed in the evening of proestrus may be critical for the establishment of a constitutive steroidogenic pathway and progesterone secretion by the corpus luteum during the diestrus stage of the murine estrous cycle.     

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by alphaMSH and ornithine decarboxylase inhibition

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by alpha-melanocyte-stimulating hormone (alphaMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, alphaMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or alphaMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor alpha-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with alphaMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to alphaMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by alphaMSH.     

Structure–activity studies on acetylcholinesterase inhibition in the lycorine series of Amaryllidaceae alkaloids

The synthesis of differentially functionalized analogs of the Amaryllidaceae alkaloid lycorine, accessed via a concise chemoselective silylation strategy, is described uncovering two of the most potent inhibitors of acetylcholinesterase (AChE) identified to date in this series. Important elements of this novel pharmacophore were elucidated through structure–activity relationship (SAR) studies.     

In vitro antiprotozoal activity of alkaloids from Phaedranassa dubia (Amaryllidaceae)

The bulbs of Phaedranassa dubia (Amaryllidaceae) were found to contain the novel compound phaedranamine, together with seven known alkaloids. The structure and stereochemistry of the alkaloids were determined by physical and spectroscopic methods. An in vitro screening against four different parasitic protozoa was carried out using the isolated compounds. The alkaloids ungeremine, pseudolycorine and haemanthamine showed good activity in in vitro assays against Trypanosoma brucei rhodesiense, T. cruzi and Plasmodium falciparum with IC₅₀ values in the range of 3.66 μM or lower.