An Agrobacterium-Mediated Transient Expression Method for Functional Assay of Genes Promoting Disease in Monocots.

Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of ß-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

Relationship between serotonin transporter gene polymorphism and constipation in cancer patients.

INTRODUCTION: To assess the potential association between serotonin transporter gene insertion/deletion polymorphism and the cancer-related constipation phenotype. MATERIAL AND METHODS: A total of 120 patients diagnosed with malignant solid tumors were subjected to genotyping. For the two groups - patients with constipation and constipation-free patients with non-gastrointestinal cancer, 60 cases in each group - we collected the peripheral venous blood. We extracted genomic DNA, and used polymerase chain reaction (PCR) to analyze the serotonin transporter (5-HT) link polymorphic region (5-HTTLPR) polymorphism of the serotonin transporter gene. RESULTS: The frequency of S/S genotype in cancer patients with constipation was 66.67% (40/60), and the frequency of the S allele was 79.17% (95/120); the frequency of S/S genotype in cancer patients without constipation was 48.33% (29/60), and the frequency of the S allele was 65.83% (79/120). There was a significant difference between the two groups (p < 0.05). CONCLUSIONS: The presence of 5-HTTLPRS/S genotype and the S allele in patients with cancers probably carry an increased risk of constipation. However, its role as a cause of cancer-related constipation needs to be further investigated.

Recombinant human brain natriuretic peptide therapy combined with bone mesenchymal stem cell transplantation for treating heart failure in rats.

This study aimed to investigate the effects of combined recombinant human brain natriuretic peptide (rhBNP) therapy and bone mesenchymal stem cell (BMSC) transplantation on cell survival in myocardial tissues and on heart function in a rat model of heart failure (HF). Rat BMSCs were isolated, amplified and adherent cultured in vitro. A rat model of HF was established via the intraperitoneal injection of doxorubicin (Adriamycin). The rats were randomly divided into normal, HF, BMSC, rhBNP and BMSC plus rhBNP groups. The BMSCs were administered once via tail vein injection and rhBNP was infused via the jugular vein. Echocardiography and polygraphy were used to evaluate heart function. An enzyme­linked immunosorbent assay was used to detect the changes in brain natriuretic peptide (BNP) concentration prior to and following intervention. Western blot analysis was used to detect the expression of the myocardium­specific proteins GATA-binding protein 4 (GATA-4), connexin 43 (Cx43) and cardiac troponin I (cTnI). The results of cardiac echocardiography and the hemodynamic data show that various indicators of left ventricular systolic function in the BMSC plus rhBNP group were significantly improved compared with those in the other groups (P<0.05). No significant differences in the improvement of cardiac function were observed between the BMSC and rhBNP groups (P>0.05). Following treatment, a significant difference in BNP levels was observed between the BMSC plus rhBNP and the BMSC groups (P<0.05). The GATA-4, Cx43 and cTnI expression levels in the BMSC plus rhBNP group were higher than those in the BMSC group. Compared with rhBNP treatment, BMSC transplantation alone does not significantly improve heart function. However, combining rhBNP therapy and BMSC transplantation increases the expression levels of GATA-4 and other proteins to improve cardiac systolic and diastolic function.

An Egr-1-specific DNAzyme regulates Egr-1 and proliferating cell nuclear antigen expression in rat vascular smooth muscle cells.

The aim of the present study was to transfect rat aortic smooth muscle cells with an early growth response factor-1 (Egr-1)-specific DNAzyme (ED5), to observe its effect on Egr-1 and proliferating cell nuclear antigen (PCNA) expression and to elucidate the mechanism of ED5-mediated inhibition of vascular smooth muscle cell (VSMC) proliferation. VSMCs in primary culture obtained by tissue block adhesion were identified by morphological observation and α smooth muscle actin (α-SM-actin) immunocytochemistry. The cells were then transfected with ED5 or scrambled ED5 (ED5SCR). The three groups of cells used in the present study were the control group, ED5 group and ED5SCR group. The expression levels of Egr-1 and PCNA protein were detected following transfection by analyzing and calculating the integral optical density value in each group. Primary culture of VSMCs and transfection of ED5 and ED5SCR were successfully accomplished. Following stimulation with 10% fetal calf serum, the Egr-1 protein was expressed most strongly at 1 h and demonstrated a declining trend over time; the expression of PCNA protein began at 4 h, peaked at 24 h and then demonstrated a slightly declining trend over time. Compared with the control group and the ED5SCR group, ED5 inhibited the expression of Egr-1 and PCNA (P<0.05). ED5 was able to inhibit the expression of Egr-1 and PCNA proteins in VSMCs to a certain extent and VSMC proliferation in vitro. DNAzyme gene therapy may be useful as a new method for treating vascular proliferative diseases, including atherosclerosis and restenosis.