Development of an improved animal model of experimental autoimmune myositis.

Multiple animal models of experimental autoimmune myositis (EAM) have been developed. However, these models vary greatly in the severity of disease and reproducibility. The goal of this study was to test whether vaccination twice with increased dose of rat myosin and pertussis toxin (PT) could induce EAM with severer disease in mice. BALB/c mice were injected with 1 mg rat myosin in 50% complete Freund's adjuvant (CFA) weekly for four times and one time of PT (EAM) or twice with 1.5 mg myosin in CFA and PT (M-EAM). In comparison with that in the CFA and PT injected controls, vaccination with rat myosin and injection PT significantly reduced the muscle strength and EMG duration, elevated serum creatine kinase levels, promoted inflammatory infiltration in the muscle tissues, leading to pathological changes in the muscle tissues, demonstrating to induce EAM. Interestingly, we found that vaccination twice with the high dose of myosin and PT prevented EAM-related gain in body weights and caused significantly less muscle strength in mice. More importantly, all of the mice receiving high dose of myosin and PT survived while 3 out of 16 mice with four times of low dose of myosin died. Finally, vaccination with high dose of myosin promoted CD4(+) and CD8(+) T cell infiltration in the muscle tissues and up-regulated MHC-I expression in the muscle tissues of mice. Hence, the new model of EAM is a time-saving, efficient and easily replicable tool for studying autoimmune myositis.

[The expression and significance of TLR4, MyD88 and NF-κB mRNA in mouse lymph node of experimental autoimmune myositis].

AIM: To investigate TLR4, MyD88 and NF-κB mRNA levels in mouse lymph node with experimental autoimmune myositis(EAM)and determine the role of TLR4 in autoimmune myositis. METHODS: Thirty femal BALB/c mice were randomly divided into five groups (n=6 animals per group): group 1 was the control, while animals in other four groups were killed at different time point: group 2 in the first week, group 3 in the second week, group 4 in the third week and group 5 in the fourth week since they had been given myosin for preparing EAM. The expressions of TLR4, MyD88 and NF-κB mRNA were measured with real-time fluorescent quantitative polymerase chain reaction. RESULTS: (1)The expressions of TLR4, MyD88 and NF-κB mRNA in each EAM group were significantly high compared with those in the normal control group, which was significantly highest in group 3 of all(P<0.01) and significantly higher in group 4 than in group 5(P<0.01).(2)The expression level of TLR4 mRNA had significant positive correlations with the expressions of MyD88 mRNA and NF-κB mRNA(r=0.906, r=0.967, P<0.01), and the latter two also had significant positive correlations(r=0.919, P<0.01). CONCLUSION: TLR4 played an important role in the development of autoimmune myositis and run its function mainly by MyD88-dependent pathway that could activate NF-κB for promoting the release of inflammatory factors.

[Construction of selectable marker-removable plant expression vectors].

The commonly used plant constitutive expression vector pBI121 was modified by insertion of two directly orientated lox sites each at one end of the selectable marker gene NPTII and by replacing the GUS gene with a sequence composed of multiple cloning sites (MCS). The resulting plant expression vector pBI121-lox-MCS is widely usable to accommodate various target genes through the MCS, and more importantly to allow the NPTII gene removed from transformed plants upon the action of the Cre recombinase. In addition, the CaMV 35S promoter located upstream of the MCS can be substituted with any other promoters to form plant vectors with expression features specified by the introduced promoters. Provided in this paper is an example that an enhanced phloem-specific promoter of the pumpkin PP2 gene (named dENP) was used to construct an NPTII-removable phloem-specific expression vector pBdENP-lox-MCS. Moreover, to facilitate screening of selectable marker-removed gene and the composite sequence is flanked by lox sites. Thus the selectable marker-free plants can be visually identified by loss of GFP fluorescence. The above newly created plant expression vectors can be used to develop selectable marker-removable transgenic plants for a variety of purposes.

A GABAergic projection from the central nucleus of the amygdala to the parabrachial nucleus: an ultrastructural study of anterograde tracing in combination with post-embedding immunocytochemistry in the rat.

To determine whether axonal terminals emanating from the central nucleus of amygdala (Ce) to the parabrachial nucleus (PBN) contain gamma-aminobutyric acid (GABA) as their neurotransmitter, an electron microscopic study was performed employing the combined techniques of WGA-HRP anterograde tracing and post-embedding immunocytochemistry for GABA. Our analysis distinguished a large population of GABA immunopositive axonal terminals from the Ce that exhibited symmetrical synaptic contacts with neurons in the lateral parabrachial nucleus. Additionally, most retrogradely labeled dendrites and perikarya received synaptic contacts from GABA immunoreactive terminals, with some of them originating from the Ce. The present study provides the first direct ultrastructural evidence for a monosynaptic, GABAergic link between Ce axons and neurons of the parabrachial nucleus via classical symmetrical synapses.

[A simple and visible assay for cre recombinase activity in Escherichia coli by using two incompatible plasmids].

The Cre/loxP system derived from bacteriophage P1 is widely used to carry out complex manipulations of DNA molecules both in vitro and in vivo. In order to further characterize and modify the Cre/loxP system, a convenient method for assaying the recombination efficiency is needed. A simple and visible assay is described, in which two incompatible plasmids, separately carrying the cre gene and loxP-flanked gfp gene, were co-transferred into E. coli. The cre gene was inserted into a kanamycin-resistant bacterial expression vector, designated pET30a-Cre. The gfp gene, flanked by directly repeated loxP sites, was cloned into an ampicillin-resistant expression vector to generate pET23b-loxGFP. E. coli BL21 (DE3) was cotransformed with pET30a-Cre and pET23b-loxGFP, and cultured in the presence of both ampicillin and kanamycin. Under UV illumination, the Cre-mediated recombination events can be easily detected. The fidelity of recombination was verified by SDS-PAGE analysis and restriction analysis followed by DNA sequencing. Thus, this cotransformation method provides a straightforward assay that can be used to modify the Cre/loxP system.

[Using green fluorescent protein as a reporter to monitor elimination of selectable marker genes from transgenic plants].

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.

Colocalization of gamma-aminobutyric acid and acetylcholine in neurons in the laterodorsal and pedunculopontine tegmental nuclei in the cat: a light and electron microscopic study.

Cholinergic and gamma-aminobutyric acid (GABA) mechanisms in the dorsolateral pontomesencephalic tegmentum have been implicated in the control of active (REM) sleep and wakefulness. To determine the relationships between neurons that contain these neurotransmitters in this region of the brainstem in adult cats, combined light and electron microscopic immunocytochemical procedures were employed. Light microscopic analyses revealed that choline acetyltransferase (ChAT) and GABA immunoreactive neurons were distributed throughout the laterodorsal and pedunculopontine tegmental nuclei (LDT and PPT). Surprisingly, approximately 50% of the ChAT immunoreactive neurons in these nuclei also contained GABA. Using electron microscopic pre-embedding immunocytochemistry, GABA immunoreactivity was observed in somas, dendrites and axon terminals in both the LDT and PPT. Most of the GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites. Electron microscopic double-immunolabeling techniques revealed that ChAT and GABA were colocalized in axon terminals in the LDT/PPT. Approximately 30% of the ChAT immunoreactive terminals were also GABA immunoreactive, whereas only 6-8% of the GABA immunoreactive terminals were ChAT immunoreactive. Most of the ChAT/GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites; however, ChAT/GABA immunoreactive terminals were also observed that contacted ChAT immunoreactive dendrites. With respect to ChAT immunoreactive postsynaptic profiles, approximately 40% of the somas and 50% of the dendrites received synaptic contact from GABA immunoreactive terminals in both the LDT and PPT. These findings (a) indicate that there are fundamental interactions between cholinergic and GABAergic neurons within the LDT/PPT that play an important role in the control of active sleep and wakefulness and (b) provide an anatomical basis for the intriguing possibility that a mechanism of acetylcholine and GABA co-release from the terminals of LDT/PPT neurons is involved in the regulation of behavioral states.