Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences

Summary With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T⁺), also had acquired non-selected calf thymus carrier DNA sequences (C⁺), being integrated in their nuclear genomes. From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas⁺), shoots were grafted and mature, flowering plants (gNT4) were obtained. After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features. The mannopine locus segregated independently from the locus determining the crown gall phenotype. When screened for integrated (transforming) foreign DNA sequences 97% of the NT4-like seedlings turned out to be C⁺T⁺. Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C⁺T⁺:C^-T⁺: C⁺T:C T SR1-like seedlings. Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes.     

Hyperammonaemia causes many of the changes found after portacaval shunting.

1. Portacaval shunting in rats results in several metabolic alterations similar to those seen in patients with hepatic encephalopathy. The characteristic changes include: (a) diminution of cerebral function; (b) raised plasma ammonia and brain glutamine levels; (c) increased neutral amino acid transport across the blood-brain barrier; (d) altered brain and plasma amino acid levels; and (e) changes in brain neurotransmitter content. The aetiology of these abnormalities remains unknown. 2. To study the degree to which ammonia could be responsible, rats were made hyperammonaemic by administering 40 units of urease/kg body weight every 12 h and killing the rats 48 h after the first injection. 3. The changes observed in the urease-treated rats were: (a) whole-brain glucose use was significantly depressed, whereas the levels of high-energy phosphates remained unchanged; (b) the permeability of the blood-brain to barrier to two large neutral amino acids, tryptophan and leucine, was increased; (c) blood-brain barrier integrity was maintained, as indicated by the unchanged permeability-to-surface-area product for acetate; (d) plasma and brain amino acid concentrations were altered; and (e) dopamine, 5-hydroxytryptamine (serotonin) and noradrenaline levels in brain were unchanged, but 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of 5-hydroxytryptamine, was elevated. 4. The depressed brain glucose use, increased tryptophan permeability-to-surface-area product, elevated brain tryptophan content and rise in the level of cerebral 5-HIAA were closely correlated with the observed rise in brain glutamine content. 5. These results suggest that many of the metabolic alterations seen in rats with portacaval shunts could be due to elevated ammonia levels. Furthermore, the synthesis or accumulation of glutamine may be closely linked to cerebral dysfunction in hyperammonaemia.     

CORRELATION OF PLASMA AND BRAIN AMINO ACID AND PUTATIVE NEUROTRANSMITTER ALTERATIONS DURING ACUTE HEPATIC COMA IN THE RAT

During acute hepatic coma following two-stage hepatic devascularization in the rat, profound changes occurred in plasma and whole-brain amino acids and putative neurotransmitters. Brain ammonia, glutamine and GABA were increased, aspartate was decreased, while glutamate was unchanged. An increase in brain tryptophan was accompanied by a similar increase in plasma unbound tryptophan but decreased plasma total tryptophan. These changes occurred in the presence of high plasma levels of the other neutral amino acids, including the branched chain amino acids. Plasma insulin was unchanged while glucagon levels rose, resulting in a decreased insulin to glucagon ratio. These results suggest that while plasma unbound tryptophan may influence brain tryptophan levels, altered plasma concentrations of neutral amino acids which compete with tryptophan for transport into the brain do not contribute to the increase in brain tryptophan observed during acute hepatic coma.     

TRYPTOPHAN TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER DURING ACUTE HEPATIC FAILURE

Abstract— Tryptophan transport across the blood-brain barrier was studied using a single injection dual isotope label technique, in the following three conditions: normal rats, rats with portacaval shunts, and rats with portacaval shunts followed 65 h later by hepatic artery ligation. In both normal rats and those with acute hepatic failure the tryptophan transport system was found to be comprised of two kinetically distinct components. One component was saturable and obeyed Michaelis-Menten kinetics (normal: V_max= 19.5 nmol.min^?1.g^?1. K_m= 113 μM; hepatic failure: V_max, = 33.8 nmol.min^?1.g^?1, K_m= 108 μM), and the second was a high capacity system which transported tryptophan in direct proportion to concentration over the range tested (normal: K= 0.026 ml.min^?1.g^?1; hepatic failure: K= 0.067 ml.min^?1.g^?1). Since the saturable low capacity component transports several neutral amino acids, and their collective plasma concentration is high in relation to the individual K_ms, tryptophan transport by this component is reduced by competitive inhibition under physiological conditions. Thus it was calculated that in normal rats approx 40% of tryptophan influx occurs via the high capacity system. During acute hepatic failure transport via both components was increased substantially, approximately doubling the rate of tryptophan penetration of the blood-brain barrier at all concentrations tested. The contribution by the high capacity component became even more significant than in normal rats, accounting for about 75% of all tryptophan passage from plasma to brain. Brain tryptophan content was 29.9 nmol/g in normal rats and rose to 45.2 nmol/g in rats with portacaval shunts and 50.5 nmol/g in those with acute hepatic failure, correlating with the increased rate of tryptophan transport. In a previous study we found that plasma competing amino acids were greatly increased during acute hepatic failure. Calculations predict that these increased concentrations would cause a reduction in tryptophan transport by the low capacity system. However, because of the increase in the rate of transport by the high capacity component, net tryptophan entry across the blood-brain barrier was actually increased. This increased rate of transport clearly contributes to the increased content of brain tryptophan found during hepatic failure.     

The mechanism of alphaIIbbeta3 antagonism by savignygrin and its implications for the evolution of anti-hemostatic strategies in soft ticks

Savignygrin, a alphaIIbbeta3 antagonist presents the RGD sequence on the substrate-binding loop of the (BPTI-fold). This study investigated whether this is the only integrin-targeting motif associated with its mechanism. It forms a tight-binding complex with alphaIIbbeta3 that is resistant to SDS dissociation under reducing and non-reducing conditions, but not to temperature or EDTA. The same complex is formed on resting and activated platelets, as well as aggregated platelets that have been disaggregated with savignygrin. Binding of FITC labeled savignygrin to platelets show that the binding kinetics and affinity of savignygrin is similar for resting and activated platelets (Kd approximately 50-70 nM). Binding to resting or activated platelets was significantly inhibited by two savignygrin peptide fragments, S2 (GSRGDEDATFG) and S3 (FDREDGGSRQG) that correspond with two specific loop-like areas in the structure of savignygrin that together form a continuous binding interface. The inability of S3 to inhibit platelet aggregation indicates that it targets a novel ligand-binding site. A model of alphaIIbbeta3 based on the recent crystal structure of alphavbeta3 into which the RGD sequence of savignygrin was docked shows that savignygrin lies along the interface formed by the two subunits. A novel mode of integrin antagonism is indicated that includes the targeting of distinct sites on the alphaIIbbeta3 subunits. The S2 and S3 loops are not involved in the mechanisms of the related soft tick blood coagulation inhibitors and suggest that this allowed their evolution as integrin targeting motifs.     

Catecholase activity associated with copper-S100B

This study addresses the spectroscopic properties and reactivity associated with the copper-loaded form of S100B isolated from bovine brain. Copper(II)-S100B displays EPR features typical of a type II copper center and is shown here to exhibit catecholase activity, the two-electron oxidation of catechols. The steady-state kinetics associated with the oxidation of several catecholamines has been probed in order to further characterize this activity. The evidence provided indicates that the catecholase chemistry is copper initiated. Superoxide dismutase has no effect on the rates of catecholamine oxidation catalyzed by Cu-S100B, establishing that superoxide is not produced during this reaction, ruling out an autoxidative mechanism. Addition of catalase to the Cu-S100B reaction with catechols reduces the amount of oxygen consumed by 50%, demonstrating that peroxide is released during this reaction. The release of peroxide is mechanistically distinct from the type III dinuclear copper proteins, catechol oxidase and tyrosinase.     

Biological activity of SeMNPV, AcMNPV, and three AcMNPV deletion mutants against Spodoptera exigua larvae (Lepidoptera: noctuidae)

Virulence and speed of action, as related to dose, are important effectiveness-determining properties of insect-pathogenic biocontrol agents. We used the droplet-feeding bioassay to compare dose responses between two wild-type baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), and three deletion mutants of AcMNPV in S. exigua larvae. In each mutant one gene was deleted by genetic engineering: pp34, coding for the polyhedral membrane; egt, coding for ecdysteroid UDP-glucosyltransferase; or p10, coding for fibrillar structures in infected insect cells. SeMNPV had the lowest median lethal dose (LD(50)) as well as the highest speed of action (LT(50)) of all viruses investigated. In our comparative bioassays the only significant effect of gene deletions in AcMNPV was a slightly lower speed of action for the p10 deletion mutant. Otherwise, wild-type and recombinant AcMNPVs had similar biological activities. Our results suggest, in contrast to what is generally assumed, that gene deletions in AcMNPV for improved insecticidal activity should be critically assessed in each host system prior to further implementation as a control agent. Insertion of foreign genes coding for entomotoxins is less questionable and more promising in this respect.     

Apyrase Activity and Platelet Aggregation Inhibitors in the Tick Ornithodoros Savignyi (Acari: Argasidae)

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activities supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.     

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.