Effects of Kv1.3 knockout on pyramidal neuron excitability and synaptic plasticity in piriform cortex of mice.

Kv1.3 belongs to the voltage-gated potassium (Kv) channel family, which is widely expressed in the central nervous system and associated with a variety of neuropsychiatric disorders. Kv1.3 is highly expressed in the olfactory bulb and piriform cortex and involved in the process of odor perception and nutrient metabolism in animals. Previous studies have explored the function of Kv1.3 in olfactory bulb, while the role of Kv1.3 in piriform cortex was less known. In this study, we investigated the neuronal changes of piriform cortex and feeding behavior after smell stimulation, thus revealing a link between the olfactory sensation and body weight in Kv1.3 KO mice. Coronal slices including the anterior piriform cortex were prepared, whole-cell recording and Ca2+ imaging of pyramidal neurons were conducted. We showed that the firing frequency evoked by depolarization pulses and Ca2+ influx evoked by high K+ solution were significantly increased in pyramidal neurons of Kv1.3 knockout (KO) mice compared to WT mice. Western blotting and immunofluorescence analyses revealed that the downstream signaling molecules CaMKII and PKCα were activated in piriform cortex of Kv1.3 KO mice. Pyramidal neurons in Kv1.3 KO mice exhibited significantly reduced paired-pulse ratio and increased presynaptic Cav2.1 expression, proving that the presynaptic vesicle release might be elevated by Ca2+ influx. Using Golgi staining, we found significantly increased dendritic spine density of pyramidal neurons in Kv1.3 KO mice, supporting the stronger postsynaptic responses in these neurons. In olfactory recognition and feeding behavior tests, we showed that Kv1.3 conditional knockout or cannula injection of 5-(4-phenoxybutoxy) psoralen, a Kv1.3 channel blocker, in piriform cortex both elevated the olfactory recognition index and altered the feeding behavior in mice. In summary, Kv1.3 is a key molecule in regulating neuronal activity of the piriform cortex, which may lay a foundation for the treatment of diseases related to piriform cortex and olfactory detection.

[Research progress in synthetic biology of active compounds in Chinese medicinal plants].

Mecicinal plants boast abundant natural compounds with significant pharmacological activity, and such compounds, featuring diversified and complex structures, can be used for research and development of drugs. At present, these natural compounds are directly extracted from herbs which, however, suffer from damaged wild resources and shortage of planting resources attributing to the increasing demand. Moreover, the low content in medicinal plants and complex structures are another challenge to the research and development of drugs. Heterologous synthesis with synthetic biology methods is a solution that has attracted wide attention. Synthetic bio-logy for the production of natural active compounds in Chinese medicinal plants involves the exploration of key enzymes in compound bio-synthetic pathways from plants, analysis of enzyme functions and mechanisms, and reconstruction and optimization of biosynthetic pathways in microorganisms for efficient synthesis of compounds. This study briefed the development process of synthetic biology and the biosynthetic pathways of terpenoids, alkaloids, and flavonoids, and summarized the related strategies of synthetic biology such as the reconstruction and optimization of metabolic pathways, regulation of fermentation process, and strain improvement, and the latest applications of heterogeneous synthetic biology in the production of natural compounds from Chinese medicinals. This study is expected to serve as a reference for the efficient production of terpenoids, alkaloids, flavonoids, and other active compounds from Chinese medicinal plants with strategies of synthetic biology.

[Cloning and identification of ROP2 gene of Toxoplasma gondii].

OBJECTIVE: To amplify ROP2 from the genomic DNA of Toxoplasma gondii RH strain and construct eukaryotic expression plasmid pc-DNA3-ROP2. METHODS: Tachyzoites of T. gondii RH strain were collected and depurated to obtain genome. A pair of primers was designed and synthesized according ROP2 gene sequence. The gene fragment encoding ROP2 was amplified from the genomic DNA of T. gondii RH strain by means of PCR. It was then reclaimed and purified, and inserted into cloning vector pUCm-T. The recon was cut by EcoR I, Hind III, and the inserted ROP2 gene fragment was subcloned into pc-DNA3 eukaryotic expression vector using T4DNA ligase, followed by further PCR identification, double digestion via restrictive enzymes, and sequencing. RESULTS: The amplified specific gene fragment of ROP2 was about 1.7 kb in length. The gene fragment cloned and subcloned into pc-DNA3 was correct, and the eukaryotic expression plasmid contained ROP2 gene fragment was successfully constructed. CONCLUSION: The recombinant plasmid pc-DNA3-ROP2 was successfully constructed.

Biological role of surface Toxoplasma gondii antigen in development of vaccine.

AIM: To analyze the biological role of the surface antigen of Toxoplasma gondii (T gondii) in development of vaccine. METHODS: The surface antigen of T gondii (SAG1) was expressed in vitro. The immune response of the host to the antigen was investigated by detection of specific antibody reaction to SAG1 and production of cytokines. Mice were immunized with recombinant SAG1 and challenged with lethal strain of T gondii RH. The monoclonal antibody to r-SAG1 was prepared and used to study the effects of SAG1 on T gondii tachyzoites under electromicroscope. RESULTS: The mice immunized with recombinant SAG1 delayed death for 60 h compared to the control group. The recombinant SAG1 induced specific high titer of IgG and IgM antibodies as well as IFN-gamma, IL-2 and IL-4 cytokines in mice. In contrast, IL-12, IL-6 and TNF-alpha were undetectable. When T gondii tachyzoites were treated with the monoclonal antibody to r-SAG1, the parasites were gathered together, destroyed, deformed, swollen, and holes and gaps formed on the surface. CONCLUSION: SAG1 may be an excellent vaccine candidate against T gondii. The immune protection induced by SAG1 against T gondii may be regulated by both hormone- and cell-mediated immune response.

[Studies on the immuno-protection of ROP2 nuclei acid vaccine in Toxoplasma gondii infection].

OBJECTIVE: To study the protective effect of ROP2 nuclei acid vaccine in mice. METHODS: Forty-two BALB/c mice were divided into three groups. Each mouse in experiment group was injected with 50 microg recombinant plasmid pc-DNA3-ROP2 through musculus quadriceps fexoris. In control groups, each mouse was injected with 50 microg blank plasmid pc-DNA3 and with 50 microl PBS respectively. All mice were immunized for three times with an interval of three weeks. The volume was doubled for the final injection in the two plasmid groups. Blood, spleens and lymph nodes of 4 mice in each group were taken for the detection of CD4+, CD8+ T cells and cytokines 2 weeks after the final immunization. The rest mice in 3 groups were challenged with 500 tachyzoites of Toxoplasm gondii RH strain for further observation. RESULTS: The vaccine induced strong cellular and humoral immune response. The titer of antibody in serum was high after inoculation and recognized ROP2 protein antigen expressed in vitro. The lymphocyte phenotype was analyzed. CD4+ T cells proliferated sharply (69.5+/-3.4)%, and the ratio of CD4/CD8+ increased considerably by (4.69+/-1.32)% (P<0.01). The level of IL-2, IL4, IL-6, IL-12, IFN-gamma and TNF in serum and cultured supernatant of spleen cells and lymph cells was higher in the experiment group than that in control groups, especially in serum. 88.9% mice in the experiment group were protected 180 hours after the challenge of T. gondii. The death time of mice in experiment group was delayed and the survival time was prolonged in comparison to that in control groups with a significant difference (P< 0.01). CONCLUSION: The recombinant ROP2 nuclei acid vaccine shows fair immunogeni-city and obviously produces immuno-protection.