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.     

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.     

Relationship between production of acute-phase proteins and strength of inflammatory stimulation in rats

The relationship between intensity of inflammatory stimulation and production of α(2)-macroglobulin (α2M) and α(1)-acid glycoprotein (AAG) in rats was investigated. Sprague-Dawley rats were injected with turpentine oil at doses of 0.05, 0.2 or 0.4 mL/rat. Serum levels of α2M, interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were measured by enzyme-linked immunosorbent assay, and AAG was measured by single radial immunodiffusion. Peak serum levels of α2M and AAG in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. However, no significant differences were observed for peak serum levels of these acute-phase proteins between 0.2 and 0.4 mL/rat. Furthermore, peak serum levels of IL-6 and CINC-1 in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. Thus, the production of these acute-phase proteins has upper limits, even under increased strength of inflammatory stimulation in rats injected with turpentine oil.     

NR4A1 (Nur77) mediates thyrotropin-releasing hormone-induced stimulation of transcription of the thyrotropin β gene: analysis of TRH knockout mice

Thyrotropin-releasing hormone (TRH) is a major stimulator of thyrotropin-stimulating hormone (TSH) synthesis in the anterior pituitary, though precisely how TRH stimulates the TSHβ gene remains unclear. Analysis of TRH-deficient mice differing in thyroid hormone status demonstrated that TRH was critical for the basal activity and responsiveness to thyroid hormone of the TSHβ gene. cDNA microarray and K-means cluster analyses with pituitaries from wild-type mice, TRH-deficient mice and TRH-deficient mice with thyroid hormone replacement revealed that the largest and most consistent decrease in expression in the absence of TRH and on supplementation with thyroid hormone was shown by the TSHβ gene, and the NR4A1 gene belonged to the same cluster as and showed a similar expression profile to the TSHβ gene. Immunohistochemical analysis demonstrated that NR4A1 was expressed not only in ACTH- and FSH- producing cells but also in thyrotrophs and the expression was remarkably reduced in TRH-deficient pituitary. Furthermore, experiments in vitro demonstrated that incubation with TRH in GH4C1 cells increased the endogenous NR4A1 mRNA level by approximately 50-fold within one hour, and this stimulation was inhibited by inhibitors for PKC and ERK1/2. Western blot analysis confirmed that TRH increased NR4A1 expression within 2 h. A series of deletions of the promoter demonstrated that the region between bp -138 and +37 of the TSHβ gene was responsible for the TRH-induced stimulation, and Chip analysis revealed that NR4A1 was recruited to this region. Conversely, knockdown of NR4A1 by siRNA led to a significant reduction in TRH-induced TSHβ promoter activity. Furthermore, TRH stimulated NR4A1 promoter activity through the TRH receptor. These findings demonstrated that 1) TRH is a highly specific regulator of the TSHβ gene, and 2) TRH mediated induction of the TSHβ gene, at least in part by sequential stimulation of the NR4A1-TSHβ genes through a PKC and ERK1/2 pathway.     

Herbivore-induced indirect defense across bean cultivars is independent of their degree of direct resistance

We tested the extent to which resistance of common bean (Phaseolus vulgaris) cultivars to the spider mite Tetranychus urticae parallels the extent to which these plants display indirect defenses via the induced attraction of the predatory mite Phytoseiulus persimilis. First, via field and greenhouse trials on 19 commercial bean cultivars, we selected two spider mite-resistant (Naz and Ks41128) and two susceptible (Akthar and G11867) cultivars and measured the spider mite-induced volatiles and the subsequently induced attraction of predatory mites via olfactory choice assays. The two major volatiles, 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and (Z)-3-hexenyl-acetate, were induced in the resistant but not in the susceptible cultivars. However, uninfested susceptible cultivars emitted these volatiles at levels similar to those of mite-infested resistant cultivars. Significant induction of several minor components was observed for all four cultivars except for the infested-susceptible cultivar G11867. Both, the spider mite-resistant cultivar Naz and the susceptible cultivar G11867, attracted more predatory mites when they were infested. In contrast, spider mites induced increased emission of two major and five minor volatiles in Ks41128, but predatory mites did not discriminate between infested and uninfested plants. Overall, the attraction of predatory mites appeared to correlate positively with the presence of TMTT and (Z)-3-hexenyl acetate and negatively with β-caryophyllene and α-pinene in the bean headspace. Taken together, our data suggest that resistance and attraction of natural enemies via induced volatiles are independent traits. We argue that it should be possible to cross predator-attraction promoting traits into resistant cultivars that lack sufficiently inducible indirect defenses.