The plastome of Citrus. Physical map,variation among Citrus cultivars and species and comparison with related genera

Summary A physical plastome map was constructed for Citrus aurantium, and the plastomes of species and cultivars of Citrus and of two Citrus relatives were analysed by Southern blot-hybridisation of labelled total tobacco cpDNA to digests of total Citrus DNA. A resemblance was found between the plastomes of cultivars of C. limon (lemon), C. sinensis (orange), C. aurantium (sour orange), C. paradisii (grapefruit) and C. grandis (pomello). The plastomes of other Citrus types such as mandarin (C. reticulata) and citron (C. medico) differed from each other as well as from the plastomes of the aforementioned group. The plastomes of Poncirus trifoliata and Microcitrus sp. are distinct from each other as well as from the Citrus types.     

Diagnosis of rotavirus, adenovirus, and herpesvirus infections by immune electron microscopy using a serum-in-agar diffusion method

The sensitivity of immune electron microscopy (IEM) for the detection and identification of bovine rotavirus, infectious bovine rhinotracheitis virus (IBR), and canine adenovirus has been studied by using the serum-in-agar (SIA) method in which a specific antiserum has been incorporated in agar.     

Variations in Lipid and Fatty Acid Content in Relation to Acetyl CoA Carboxylase in the Marine Prymnesiophyte Isochrysis galbana

Lipid and fatty acid content was determined in Isochrysis galbanacultures grown under various environmental conditions in steadystate continuous cultures. Lipid and fatty acid accumulationwas observed under severe nitrogen limiting conditions. Therate of in situ lipid synthesis, determined from ¹⁴C bicarbonateincorporation into lipid fraction, decreased under nitrogenlimited growth (µ<0.72day^–1), concomitant witha reduction in the in vitro activity of the enzyme acetyl CoAcarboxylase. Cellular lipid and fatty acid content remainedfairly constant over a wide range of irradiance levels. Therate of lipid synthesis, however, increased as irra-diance levelwas elevated to a maximal value at a light intensity of 150µmolquanta m^–2s^–1 and slightly decreased at a higherphoton flux. The in vitro activity of ACCase roughly followedthe same pattern of lipid synthesis in response to light intensity.The relative abundance of acetyl CoA carboxylase significantlydecreased in nitrogen limited cultures grown at low dilutionrates (µ<0.72day^–1). A fairly good linear correlationwas measured between the cellular content of ACCase and theenzyme activity in cultures grown under nitrogen limiting conditions.Furthermore, in nitrogen limited cultures, the cellular fattyacid content was linearly related to the cell capacity to producemalonyl CoA, the end product of ACCase and the building blockin fatty acid synthesis. (Received November 20, 1990; Accepted January 17, 1991)     

The protective effects of Prunus armeniaca L (apricot) against methotrexate-induced oxidative damage and apoptosis in rat kidney

This study was conducted to evaluate a possible protective role of apricot in apoptotic cell death induced by methotrexate (MTX) and renal damage by different histological and biochemical parameters. Twenty-eight rats were divided into four groups, control, apricot, methotrexate, and apricot + methotrexate. Methotrexate induced renal failure, as shown by significant serum creatinine and urea elevation. Additionally, the results indicated that methotrexate significantly induced lipid peroxidation and reduced antioxidant activities in rats. In contrast, apricot significantly prevented toxic effects of methotrexate via increased catalase, superoxide dismutase, and glutathione levels but decreased formation of malondialdehyde. Also, it was determined that exposure to methotrexate leads to significant histological damage in kidney tissue such as glomerulosclerosis and apoptosis. On the other hand, these effects can be eliminated with apricot diet. These data indicate that apricot may be useful in preventing undesirable effects of MTX such as nephrotoxicity.     

Optimized small‐molecule pull‐downs define MLBP1 as an acyl‐lipid‐binding protein

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand‐binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC‐MS to identify candidate binding ligands. We optimized this method using ABA–PYL interactions and show that ABA co‐purifies with wild‐type PYL5 but not a binding site mutant. The K_d of PYL5 for ABA is 1.1 μm , which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37 START domain‐related proteins, which resulted in the identification of ligands that co‐purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co‐purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants.     

Towards clarification of the biological role of microcystins, a family of cyanobacterial toxins

Microcystins constitute a serious threat to the quality of drinking water worldwide. These protein phosphatase inhibitors are formed by various cyanobacterial species, including Microcystis sp. Microcystins are produced by a complex microcystin synthetase, composed of peptide synthetases and polyketide synthases, encoded by the mcyA-J gene cluster. Recent phylogenetic analysis suggested that the microcystin synthetase predated the metazoan lineage, thus dismissing the possibility that microcystins emerged as a means of defence against grazing, and their original biological role is not clear. We show that lysis of Microcystis cells, either mechanically or because of various stress conditions, induced massive accumulation of McyB and enhanced the production of microcystins in the remaining Microcystis cells. A rise in McyB content was also observed following exposure to microcystin or the protease inhibitors micropeptin and microginin, also produced by Microcystis. The extent of the stimulation by cell extract was strongly affected by the age of the treated Microcystis culture. Older cultures, or those recently diluted from stock cultures, hardly responded to the components in the cell extract. We propose that lysis of a fraction of the Microcystis population is sensed by the rest of the cells because of the release of non-ribosomal peptides. The remaining cells respond by raising their ability to produce microcystins thereby enhancing their fitness in their ecological niche, because of their toxicity.     

Live-Attenuated Lentivirus Immunization Modulates Innate Immunity and Inflammation while Protecting Rhesus Macaques from Vaginal Simian Immunodeficiency Virus Challenge

Immunization with attenuated lentiviruses is the only reliable method of protecting rhesus macaques (RM) from vaginal challenge with pathogenic simian immunodeficiency virus (SIV). CD8(+) lymphocyte depletion prior to SIVmac239 vaginal challenge demonstrated that a modest, Gag-specific CD8(+) T cell response induced by immunization with simian-human immunodeficiency virus 89.6 (SHIV89.6) protects RM. Although CD8(+) T cells are required for protection, there is no anamnestic expansion of SIV-specific CD8(+) T cells in any tissues except the vagina after challenge. Further, SHIV immunization increased the number of viral target cells in the vagina and cervix, suggesting that the ratio of target cells to antiviral CD8(+) T cells was not a determinant of protection. We hypothesized that persistent replication of the attenuated vaccine virus modulates inflammatory responses and limits T cell activation and expansion by inducing immunoregulatory T cell populations. We found that attenuated SHIV infection decreased the number of circulating plasmacytoid dendritic cells, suppressed T cell activation, decreased mRNA levels of proinflammatory mediators, and increased mRNA levels of immunoregulatory molecules. Three days after SIV vaginal challenge, SHIV-immunized RM had significantly more T regulatory cells in the vagina than the unimmunized RM. By day 14 postchallenge, immune activation and inflammation were characteristic of unimmunized RM but were minimal in SHIV-immunized RM. Thus, a modest vaccine-induced CD8(+) T cell response in the context of immunoregulatory suppression of T cell activation may protect against vaginal HIV transmission.     

An acidic amino acid transmembrane helix 10 residue conserved in the neurotransmitter:sodium:symporters is essential for the formation of the extracellular gate of the γ-aminobutyric acid (GABA) transporter GAT-1

GAT-1 mediates transport of GABA together with sodium and chloride in an electrogenic process enabling efficient GABAergic transmission. Biochemical and modeling studies based on the structure of the bacterial homologue LeuT are consistent with a mechanism whereby the binding pocket is alternately accessible to either side of the membrane and which predicts that the extracellular part of transmembrane domain 10 (TM10) exhibits aqueous accessibility in the outward-facing conformation only. In this study we have engineered cysteine residues in the extracellular half of TM10 of GAT-1 and probed their state-dependent accessibility to sulfhydryl reagents. In three out of four of the accessible cysteine mutants, the inhibition of transport by a membrane impermeant sulfhydryl reagent was diminished under conditions expected to increase the proportion of inward-facing transporters, such as the presence of GABA together with the cotransported ions. A conserved TM10 aspartate residue, whose LeuT counterpart participates in a "thin" extracellular gate, was found to be essential for transport and only the D451E mutant exhibited residual transport activity. D451E exhibited robust sodium-dependent transient currents with a voltage-dependence indicative of an increased apparent affinity for sodium. Moreover the accessibility of an endogenous cysteine to a membrane impermeant sulfhydryl reagent was enhanced by the D451E mutation, suggesting that sodium binding promotes an outward-facing conformation of the transporter. Our results support the idea that TM10 of GAT-1 lines an accessibility pathway from the extracellular space into the binding pocket and plays a role in the opening and closing of the extracellular transporter gate.