Micromonospora is a normal occupant of actinorhizal nodules

Actinorhizal plants have been found in eight genera belonging to three orders (Fagales, Rosales and Cucurbitales). These all bear root nodules inhabited by bacteria identified as the nitrogen-fixing actinobacterium Frankia. These nodules all have a peripheral cortex with enlarged cells filled with Frankia hyphae and vesicles. Isolation in pure culture has been notoriously difficult, due in a large part to the growth of fast-growing contaminants where, it was later found, Frankia was slow-growing. Many of these contaminants, which were later found to be Micromonospora, were obtained from Casuarina and Coriaria. Our study was aimed at determining if Micromonospora were also present in other actinorhizal plants. Nodules from Alnus glutinosa, Alnus viridis, Coriaria myrtifolia, Elaeagnus x ebbingei, Hippophae rhamnoides, Myrica gale and Morella pensylvanica were tested and were all found to contain Micromonospora isolates. These were found to belong to mainly three species: Micromonospora lupini, Micromonospora coriariae and Micromonospora saelicesensis. Micromonospora isolates were found to inhibit some Frankia strains and to be innocuous to other strains.     

Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s

Candida apicola belongs to a group of yeasts producing surface-active glycolipids consisting of sophorose and long-chain (ω)- or (ω-1)-hydroxy fatty acids. Hydroxylation of the fatty acids in this strain is likely catalyzed by cytochrome P450 monooxygenases (P450), which require reducing equivalents delivered via a cytochrome P450-diflavin reductase (CPR). We herein report cloning and characterization of the cpr gene from C. apicola ATCC 96134. The gene encoding a protein of 687 amino acids was cloned in Escherichia coli and the enzyme was expressed in functional form after truncation of its N-terminal putative membrane anchor. The truncated recombinant protein showed cytochrome c reducing activity (K _M of 13.8 μM and k _cat of 1,915 per minute). Furthermore, we herein demonstrate to our best knowledge for the first time the use of a eukaryotic CPR to transfer electrons to bacterial P450s (namely CYP109B1 and CYP154E1). Cloning and characterization of this CPR therefore is not only an important step in the study of the P450 systems of C. apicola, but also provides a versatile redox partner for the characterization of other bacterial P450s with appealing biotechnological potential. The GenBank accession number of the sequence described in this article is JQ015264.     

Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors

GABA_A receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α₅ has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α₅ subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α₅ subunit-containing GABA_A receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α_4/6 subunit-containing GABA_A receptors, but is sensitive to L-655708, an antagonist of the GABA_A receptors containing α₅ subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α₅ subunit in the turtle spinal cord. Together, these results suggest that GABA_A receptors containing the α₅ subunit mediate the tonic inhibitory currents observed in VHIs.     

Application of a high-throughput fluorescent acetyltransferase assay to identify inhibitors of homocitrate synthase

Homocitrate synthase (HCS) catalyzes the first step of l-lysine biosynthesis in fungi by condensing acetyl-coenzyme A and 2-oxoglutarate to form 3R-homocitrate and coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of approximately 41,000 small molecules. Following confirmation, counter screens, and dose–response analysis, we prioritized more than 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases, and enzymes involved in lipid metabolism.     

Palm Management in South America

We reviewed information on management of useful palms in South America. We documented management for 96 species, from incidental activities intended to increase populations of wild palms to the inclusion of palms in complex agroforestry systems. Two species, Bactris gasipaes and Parajubaea cocoides, are domesticated. Managed species are remarkably fewer than species used in the region, which suggests that harvesters often disregard the fate of the species they use. The best way of managing palms is to employ harvest methods that do not decimate the populations. Although a variety of harvesting techniques have been documented, overharvest is common, and mismanagement prevails – unnecessary felling of palms in order to harvest leaves or fruits is a widespread practice. Research should focus on assessing production in response to management practices, but eradicating the habit of destructive harvest is an obvious priority. Research on palm management must be combined with actions addressed to all stakeholders of the palm/humans system.     

Polymer-mediated cyclodehydration of alditols and ketohexoses

The polymer PEDOT⁺ (1 or 2) mediates a cyclodehydration reaction with alditols 3, 5, 7, 9, in hydrocarbon solvents, to give cyclic ethers 4, 6, 8, or 10, respectively, in high yield with a trivial isolation protocol. Polymers 1 or 2 also mediate the cyclodehydration of ketohexoses such as d-fructose, but not aldohexoses, to the important industrial intermediate 5-hydroxymethylfurfural (17), under milder conditions when compared to reactions mediated by mineral acids. A cascade reaction with ketohexoses is observed in toluene via cyclodehydration followed by Friedel–Crafts alkylation of the initially formed benzylic alcohol to give 16.     

Prostaglandin‐endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep

Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin‐endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptus‐derived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR‐Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2‐derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally. Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2‐derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep.     

Antioxidant activity of in vitro plantlets and callus cultures of Randia echinocarpa,a medicinal plant from northwestern Mexico

Randia echinocarpa, an endemic plant to Northwest Mexico, is used as food and in traditional medicine, and several of its biological activities have been demonstrated (antioxidant, antimutagenic, antidiabetic, and immunomodulatory). Plant tissue culture is a safe and scalable system for plant propagation and production of bioactive compounds. Therefore, this study aims to establish protocols for seed germination and callus culture of R. echinocarpa and to evaluate the antioxidant activity of methanol extracts (ME) of plantlets and calli via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) methods. Seeds were cultured in media with different concentrations of Murashige and Skoog (MS) salts and sucrose, and a higher germination rate and plantlet growth was observed in half-strength MS medium with 15 g L⁻¹ of sucrose. Calli were obtained from cotyledon and hypocotyl explants cultured in MS media with different concentrations of benzyl aminopurine (BAP) and indole-3-acetic acid (IAA). All treatments induced callus formation in 100% of explants; however, the medium containing 1 mg L⁻¹ BAP + 1 mg L⁻¹ IAA was selected because it produced calli with higher biomass and friable texture. The ME of cotyledons showed the highest antioxidant activity values (μmol Trolox per 100 g dry weight) in DPPH (345.5) and ABTS (1166.4) assays, whereas the ME of calli from hypocotyls showed a higher antioxidant activity than the ME of calli from cotyledons in both antioxidant assays. The tissue culture protocols established here will be useful for R. echinocarpa germplasm conservation and propagation, as well as for the production of bioactive compounds.

     

Maintaining historic disturbance regimes increases species' resilience to catastrophic hurricanes

As habitat loss and fragmentation, urbanization, and global climate change accelerate, conservation of rare ecosystems increasingly relies on human intervention. However, any conservation strategy is vulnerable to unpredictable, catastrophic events. Whether active management increases or decreases a system's resilience to these events remains unknown. Following Hurricane Irma's landfall in our habitat restoration study sites, we found that rare ecosystems with active, human‐imposed management suffered less damage in a hurricane's path than unmanaged systems. At the center of Irma's landfall, we found Croton linearis' (a locally rare plant that is the sole host for two endangered butterfly species) survival and population growth rates in the year of the hurricane were higher in previously managed plots than in un‐managed controls. In the periphery of Irma's circulation, the effect of prior management was stronger than that of the hurricane. Maintaining the historical disturbance regime thus increased the resilience of the population to major hurricane disturbance. As climate change increases the probability and intensity of severe hurricanes, human management of disturbance‐adapted landscapes will become increasingly important for maintaining populations of threatened species in a storm's path. Doing nothing will accelerate extinction.