Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

Two isoforms of acid phosphatase secreted by tobacco protoplasts: differential effect of brefeldin A on their secretion

Summary The effects of brefeldin A (BFA) on the secretion of acid phosphatase (APase) by tobacco protoplasts were investigated. Secretion of APase was inhibited by BFA in a dose-dependent manner, with a concomitant intracellular accumulation of the enzyme. The secreted APase was composed of two isoforms. BFA (10/ g/ml) inhibited the secretion of one of the isoforms without inhibiting that of the other, and this phenomenon explains the partial inhibition of APase secretion as a whole. The inhibition of APase secretion was accompanied by changes in the morphology of the Golgi apparatus and also by an increment in massdensity of cells.Abbreviations APase acid phosphatase - BFA brefeldin A - CHX cycloheximide - PAGE polyacrylamide gel electrophoresis     

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective

To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs).

Methods

This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009.

Results

Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset.

Conclusion

Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”     

Synergistic effects of anacardic acids and methicillin against methicillin resistant Staphylococcus aureus

The synergistic effects of 6-alk(en)ylsalcylic acids, also known as anacardic acids, in combination with methicillin against Staphylococcus aureus ATCC 33591 (MRSA) was investigated. The double bond in C15-anacardic acids is not essential in eliciting the antibacterial activity but is associated with increasing the activity. The synergistic effects decreased with increasing the number of double bonds in the alkyl chain. On the other hand, the antibacterial activity of anacardic acids possessing different alkyl chain lengths against the same MRSA strain was found to be a parabolic function of their lipophilicity and maximized with the alkyl chain length of C10 and C12. Notably, the synergistic effects were noted to increase with increasing the alkyl chain length.     

TG1 integrase-based system for site-specific gene integration into bacterial genomes

Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ～2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ～10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ～10-kbp DNA into the genome with an efficiency of ～10⁴ transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli “centromere” analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC–migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.     

Polyamines and jasmonic acid induce plasma membrane potential variations in lima bean

Exogenous polyamines [cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm)] elicit the production of volatiles in Lima bean (Phaseolus lunatus). Among the tested PAs, Spm induces the production of some volatile terpenoids that are known to be induced by the spider mite Tetranychus urticae. Spm treatment elicits the biosynthesis of Jasmonic acid (JA), a phytohormone known to regulate the production of the volatile terpenoids. The treatment with JA together with Spm resulted in the increased volatile emission, and predatory mites Phytoseiulus persimilis preferred JA and Spm-treated leaves over those treated with JA alone.⁵ JA and Spm treatment has no effects on polyamine oxidase (PAO) and Cu-amine oxidase (CuAO) but has a significant induction of calcium influx, ROS production, enzyme activities for NADPH-oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase, and gene expressions except for NADPH-oxidase complex.⁵ Here, we report that a plasma membrane potential (V_m) depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd. JA perfusion did not alter V_m but the perfusion of JA and the polyamines significantly increased Cad and Put V_m depolarization. When JA was perfused with polyamines, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Key words: polyamines, lima bean, herbivore-induced volatile organic compounds, calcium and ROS signalling, jasmonic acid, quantitative gene expression, transmembrane potentialPolyamines are involved in plants’ stress responses and growth. By activating biosynthesis of nucleic acids, polyamines concern the plant growth and differentiation.^1–3 Furthermore, it has been reported that polyamines are involved in the response against environmental stress and plant disease.^1–4 We recently reported that exogenously applied polyamines ∼diamines [cadaverine (Cad), putrescine (Put)], triamine [spermidine (Spd)] and tetraamine ]spermine (Spm)]∽ induce volatile emission in Lima bean leaves.⁵ Membrane potentials (V_m) and intracellular calcium variations were also studied in Lima bean leaves after perfusion with the polyamines and with these addition of JA and here we report on these additional results.The primary candidate for intercellular signaling in higher plants is the stimulus-induced change in V_m.⁶ The plasma membrane potential (V_m), which lies in the range of −50 to −200 mV in Lima bean leaves,⁷ may be shifted either to more negative (hyperpolarization) or to more positive values (depolarization) in response to various biotic or abiotic stresses.Measurement of V_m were performed and data statistically treated as previously described (ANOVA and Tukey-Kramer’s HSD test).⁷ Perfusion with the polyamines (Fig. 1 single arrow) shows a specific response of the leaf tissues with a different V_m depolarization, depending on the polyamine. In general, a V_m depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd (Fig. 1). Spm and Spd V_m depolarization values were significantly different (p < 0.05) from all other polyamines, whereas no significant difference was found between Put and Cad V_m depolarization (p = 0.435). In all cases, V_m depolarization was reversed by washing polyamine-treated leaves with a fresh buffer solution (Fig. 1 double arrow); however, a full recovery of the V_m was observed only for Put (Fig. 1). The linearization of the data from Figure 1 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines which was higher for Spd (6.0 mV min⁻¹; R = 0.96), equal for Put and Cad (4.8 mV min⁻¹; Put R = 0.95; Cad R = 0.97) and lower for Spm (3.0 mV min⁻¹; R = 0.96).Open in a separate windowFigure 1Effect of 1 mM polyamines (arrow) on the V_m of Lima bean palisade cells. Spermine (Spm) caused the lowest V_m depolarization, whereas spermidine (Spd) showed the highest values of V_m depolarization. intermediate values were found when putrescine (Put) and cadaverine (cad) were perfused. after washing the tissues with fresh buffer (double arrow) V_m was always hyperpolarized, however the initial potential was recovered only for Put, while for all other polyamines the V_m never reached the initial values. Metric bars indicate standard deviation.Perfusion with JA caused a slight and not significant (p = 0.332) V_m depolarization (Fig. 2) with respect to control. The addition of JA caused a significant increase (p < 0.01) in V_m depolarization when perfused with Cad, with respect to the sole perfusion with Cad (Fig. 1). The same was observed when JA was perfused with Put, whereas not significant differences were observed when Spm (p = 0.513) and Spd (p = 0.107) were perfused with JA (Fig. 2), with respect to the sole perfusion with Spm and Spd (Fig. 1). The linearization of the data from Figure 2 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines + JA, which was higher for Cad (24.40 mV min⁻¹; R = 0.99), almost equal for Put and Spd (Put: 14.21 mV min⁻¹, R = 0.99; Spd: 13.49 mV min⁻¹, R = 0.99) and lower for Spm (1.34 mV min⁻¹; R = 0.93). For JA the rate of V_m depolarization was 0.19 mV min⁻¹ (R = 0.96). With the addition of JA, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Open in a separate windowFigure 2Effect of 1 mM polyamines + 0.1 mMJA (arrow) on the V_m of Lima bean palisade cells. the perfusion with Ja did not cause any variation in the V_m. addition of JA to Spm and Spd caused the same V_m depolarization observed in the absence of JA, whereas when JA was added to Put and Cad a stronger and significantly different V_m depolarization was observed. even in this case washing the tissues with fresh buffer (double arrow) caused a V_m hyperpolarized, however in this case Spd reached V_m values significantly more negative that the initial V_m. Metric bars indicate standard deviation. For abbreviations see Figure 1.Since ion fluxes through channels directly influence V_m, it seems reasonable to assume that molecules able to act on channel activity might be considered as important factors inducing electrical signals. Among the various channels, calcium and potassium channels are predominantly involved in cell signaling.⁸ In the present study, rapid and reversible V_m depolarization observed upon perfusion of Lima bean mesophyll cells with polyamines was found to be significantly increased when JA was added to Cad and Put. The reversibility of the V_m may be linked to the overall physico-chemical amphiphilic properties of polyamines, probably depending on non covalent interaction with plasma membrane molecules, as polyamines occur in plants in free form, bound electrostatically to negatively charged molecules, and conjugated to small molecules and proteins.⁹ Liu et al.¹⁰ showed that Spm, Spd, Cad and Put strongly inhibited opening and closing of stomata in Vicia faba, suggesting that polyamines target inward potassium channels in guard cells and modulate stomatal movements, so providing a link between abiotic stress, polyamine levels and stomatal regulation. Moreover, the transport of polyamines across the plasma membrane of plant cells is energy-dependent and calcium is involved in the uptake mechanism.^1,11 Both mechanisms can be correlated to the observed V_m depolarization, and the positive correlation between intracellular Ca²⁺ concentration⁵ and V_m depolarizing activity of polyamines confirms the involvement of Ca²⁺ during polyamine uptake.¹¹     

The composite effect of transgenic plant volatiles for acquired immunity to herbivory caused by inter-plant communications

A blend of volatile organic compounds (VOCs) emitted from plants induced by herbivory enables the priming of defensive responses in neighboring plants. These effects may provide insights useful for pest control achieved with transgenic-plant-emitted volatiles. We therefore investigated, under both laboratory and greenhouse conditions, the priming of defense responses in plants (lima bean and corn) by exposing them to transgenic-plant-volatiles (VOCos) including (E)-β-ocimene, emitted from transgenic tobacco plants (NtOS2) that were constitutively overexpressing (E)-β-ocimene synthase. When lima bean plants that had previously been placed downwind of NtOS2 in an open-flow tunnel were infested by spider mites, they were more defensive to spider mites and more attractive to predatory mites, in comparison to the infested plants that had been placed downwind of wild-type tobacco plants. This was similarly observed when the NtOS2-downwind maize plants were infested with Mythimna separata larvae, resulting in reduced larval growth and greater attraction of parasitic wasps (Cotesia kariyai). In a greenhouse experiment, we also found that lima bean plants (VOCos-receiver plants) placed near NtOS2 were more attractive when damaged by spider mites, in comparison to the infested plants that had been placed near the wild-type plants. More intriguingly, VOCs emitted from infested VOCos-receiver plants affected their conspecific neighboring plants to prime indirect defenses in response to herbivory. Altogether, these data suggest that transgenic-plant-emitted volatiles can enhance the ability to prime indirect defenses via both plant-plant and plant-plant-plant communications.     

Aglycon specificity profiling of alpha-glucosidases using synthetic probes

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of alpha-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae alpha-glucosidase among all alpha-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae alpha-glucosidase with Ki 0.13 mM.