Gene expression profile analysis of tobacco leaf trichomes.

BACKGROUND: Leaf trichomes of Nicotiana tabacum are distinguished by their large size, high density, and superior secretion ability. They contribute to plant defense response against biotic and abiotic stress, and also influence leaf aroma and smoke flavor. However, there is limited genomic information about trichomes of this non-model plant species. RESULTS: We have characterized Nicotiana tabacum leaf trichome gene expression using two approaches. In the first, a trichome cDNA library was randomly sequenced, and 2831 unique genes were obtained. The most highly abundant transcript was ribulose bisphosphate carboxylase (RuBisCO). Among the related sequences, most encoded enzymes involved in primary metabolism. Secondary metabolism related genes, such as isoprenoid and flavonoid biosynthesis-related, were also identified. In the second approach, a cDNA microarray prepared from these 2831 clones was used to compare gene expression levels in trichome and leaf. There were 438 differentially expressed genes between trichome and leaves-minus-trichomes. Of these, 207 highly expressed genes in tobacco trichomes were enriched in second metabolic processes, defense responses, and the metabolism regulation categories. The expression of selected unigenes was confirmed by semi-quantitative RT-PCR analysis, some of which were specifically expressed in trichomes. CONCLUSION: The expression feature of leaf trichomes in Nicotiana tabacum indicates their metabolic activity and potential importance in stress resistance. Sequences predominantly expressed in trichomes will facilitate gene-mining and metabolism control of plant trichome.

Influence of high-carbon basal fertiliser on the structure and composition of a soil microbial community under tobacco cultivation.

Soil microorganisms play a crucial role in cycling soil nutrients and providing organic nutrients for plant growth and development. Fertilisation balances soil fertility and quality, and affects soil microbial communities. Fertilisation is a frontier subject in agricultural and environmental sciences. Here we showed that the application of high-carbon basal fertiliser treatment could improve the tobacco yield and quality when compared to chemical fertiliser, high-carbon basal fertiliser and mixed high-carbon chemical fertiliser. The potential reason is that different fertiliser treatments influence soil fertility, such as nitrogen, phosphorus, and other contents, besides soil organic matter. Further experiments revealed that populations of bacteria, fungi and actinomycetes fluctuated during tobacco development under different fertilisation treatments. Then we performed high-throughput sequencing of the 16S rRNA gene, and the results showed that the fertilisation treatments had significant effects on the microbial community, particularly within the finer taxonomic divisions or non-dominant taxa. Moreover, proteobacteria and fungal genera had significantly different relative abundances during tobacco growth under various tobacco developmental stages and fertilisation treatments. These results indicated that mixed high-carbon chemical fertiliser could improve soil fertility by influencing the soil microorganism, and that the fertilisation treatments impacted on the structure and composition of the microbial community, and especially the diversity of non-dominant taxa. However, more studies are needed to confirm their reliability.

Overexpression of Tobacco GCN2 Stimulates Multiple Physiological Changes Associated With Stress Tolerance.

General control non-derepressible-2 (GCN2) is a ubiquitous protein kinase that phosphorylates the α subunit of the eukaryotic initiation factor, eIF2, preventing the initiation of a new cycle of protein synthesis, subsequently reducing the global protein biosynthesis. GCN2 can also regulate the response of plants to biotic and abiotic stresses. In this study, two GCN2 homologs, NtGCN2-1 and NtGCN2-2, were cloned from Nicotiana tabacum, and were predicted to have been derived from their progenitors in N. tomentosiformis and N. sylvestris, respectively. The phosphorylation of NteIF2α could be activated by promoting the expression of NtGCN2 with plant hormones, including salicylic acid (SA), azelaic acid (AZA), methyl jasmonate (MeJA), and by imposition of different stresses (Bemisia tabaci infection, drought, and cold), indicating that NtGCN2 is involved in the response of plants to multiple biotic and abiotic stresses. We also observed that the overexpression of NtGCN2-1 significantly influenced different physiological processes. It promoted seed germination and root elongation. The content of total soluble sugars and reducing sugars were decreased, whereas those of chlorophyll a and b were increased in the GCN2 overexpressing plants. In addition, the overexpressing plants had lower content of reactive oxygen species and exhibited higher antioxidant activities. These physiological alterations could be attributed to the changes in the endogenous phytohormones, decrease in the SA and abscisic acid content, and accumulation of MeJA and AZA. It indicated that the overexpression of NtGCN2 in tobacco, stimulated the plant defense responses via phosphorylation of NteIF2α and regulation of plant hormones, and changes in the antioxidant ability and plant nutrient status.

H-phosphonate-mediated sulfonylation of heteroaromatic N-oxides: a mild and metal-free one-pot synthesis of 2-sulfonyl quinolines/pyridines.

A smart H-phosphonate-mediated synthetic strategy for the sulfonylation of heteroaromatic N-oxides has been developed, by which a large variety of 2-sulfonyl quinolines/pyridines were synthesized starting from easily available sulfonyl chlorides, diisopropyl H-phosphonate and pyridine/quinoline N-oxides in one pot under metal-free conditions at room temperature.

[Altered gene expression of lymphatic endothelial cells in oral cancer].

OBJECTIVE: To explore altered molecular phenotype of lymphatic endothelial cells (LEC) induced by oral cancer cells. It is relevant to develop therapeutic strategies for blocking oral cancer spread through lymphatic vessels. METHODS: LEC were co-cultured with oral cancer cells in a model. Differential gene expression profiles between tumor-derived lymphatic endothelial cell (TLEC) and LEC were analyzed by Human Genome U133 Plus 2.0 GeneChip arrays. Differential expression genes of functional similarity were classified. RESULTS: Differential gene expression profiles revealed that 677 unique genes had at least a twofold change in expression level between the two groups, 384 were overexpressed, and 293 were underexpressed in TLEC. These genes were related to cell adhesion, apoptosis, cell motility, development, and angiogenesis. In addition, some genes were involved in signal transduction, immune response, cell metabolism, and so on. CONCLUSION: LEC and TLEC are distinct at the molecular level. A novel therapeutic strategy of lymphatic metastasis is encouraging and anticipated based upon manipulation of LEC responses to oral cancer cells.

[An experimental study on recombinant adenovirus p53 transfected in oral dysplastic epithelial cells].

OBJECTIVE: To investigate and evaluate the appropriate virus titer and transfection efficiency of recombinant adenovirus p53 into the oral dysplastic epithelial cells (POE-9n) and provide reference for oral precancerosis research. METHODS: The transfection sensitivity of adenovirus into oral dysplastic epithelial cells was evaluated by the recombinant adenovirus p53 containing green fluorescent protein (rAd-GFP). Different titre rAd -p53 was transfected into oral dysplastic epithelial cells to evaluate the effects of rAd-p53 on cell proliferation inhibition by MIT assay. The expression of exogenous p53 gene in POE-9n cells was detected by immunocytochemistry. RESULTS: More than 95% POE-9n cells were transfected by rAd-GFP with MOI from 100 to 500 and there was no statistical difference between different MOI values (r=-0.124, P>0.05). It was found that rAd-p53 had significant inhibition effects on POE-9n cell proliferation with MOI from 100 to 500, and there were no significant differences at 96 h and 120 h after the transfection on cell proliferation inhibition (P>0.05). P53 protein was well expressed in rAd-p53 transfected POE-9n cells. CONCLUSION: Exogenous p53 can be successfully transfected into POE-9n cells by rAd-p53 and the virus titer of MOI 100 was high enough to ensure efficient transfection.

In vitro and clinical studies of gene therapy with recombinant human adenovirus-p53 injection for oral leukoplakia.

PURPOSE: Oral leukoplakia is a well-recognized precancerous lesion of squamous cell carcinoma. When accompanied with abnormal p53 expression, it suffered a higher risk of canceration. The present study was carried out to test whether the recombinant human adenovirus-p53 could introduce wild-type p53 gene to oral leukoplakia cells and induce cell cycle arrest and apoptosis. EXPERIMENTAL DESIGN: We select p53(-) oral dysplastic keratinocyte POE-9n, to observe the growth inhibition, cell cycle change, apoptosis-induced effects, and elaborate the corresponding molecular mechanism of recombinant adenovirus-p53 on POE-9n cells. Meanwhile, we evaluate the feasibility, safety, and biological activity of multipoints intraepithelial injections of recombinant adenovirus-p53 in 22 patients with dysplastic oral leukoplakia. RESULTS: Exogenous p53 could be successfully transduced into POE-9n cells by recombinant adenovirus-p53. The optimal infecting titer in this study was multiplicity of infection (MOI) = 100. Recombinant adenovirus-p53 could strongly inhibit cell proliferation, induce apoptosis, and arrest cell cycle in stage G(1) in POE-9n cells by inducing p21(CIP/WAF) and downregulating bcl-2 expression. In the posttreatment patients, p53 protein and p21(CIP/WAF) protein expression were significantly enhanced, yet bcl-2 protein presented low expression. Sixteen patients showed clinical response to the treatment, and 14 patients showed obvious histopathologic improvement. CONCLUSION: Intraepithelial injections of recombinant human adenovirus-p53 were safe, feasible, and biologically active for patients with dysplastic oral leukoplakia.

[Biologic analysis of recombinant human adenovirus-p53 injection in patients with oral leukoplakia ].

OBJECTIVE: Advances in tumor biology and clinical trials indicate that p53 transfer is an alternative therapy for head and neck squamous cell carcinoma. The aim of this study is to evaluate the biologic activity of multiple intraepithelial injections of Ad-p53 in patients with dysplastic oral leukoplakia, which is the most common premalignant lesion of the oral squamous cell carcinoma. METHODS: From 2006 to 2007, 18 Chinese patients clinically and histopathologically diagnosed as dysplastic oral leukoplakia were recruited for this study. On a 15-day cycle, intraepithelial injections of Ad-p53 were administered once every three days at dose levels dependent upon lesion size/dose escalation sequence (1 x 10(8) vp). 24-48 h after the last injection, incisional biopsy were performed, and immunohistochemistry was used to examine the protein expression of P53 and P21(CIP/WAF). RESULTS: In the postreatment patients, P53 protein and P21(CIP/WAF) protein expression were significantly enhanced (100%, 89.9%, respectively) and statistical analysis revealed the expression of P53 protein had a positive correlation with that of P21(CIP/WAF) protein (r=0.598, P<0.01). CONCLUSION: Intraepithelial injections of gendicine is biologically active in patients with dysplastic oral leukoplakia. It may be a promising treatment for oral leukoplakia.

[The study of apoptosis induction effect of vitamin E succinate on Tca8113 human tongue cancer cells].

OBJECTIVE: To investigate the apoptosis induction effect of vitamin E succinate (VES) on Tca8113 cells and its possible mechanisms. METHODS: The proliferative activity of Tca8113 was assessed by methyl thiazolyl tetrazolium (MTT) assay. After Tca8113 cells were treated with different concentrations of VES, apoptotic rates were analyzed by flow cytometry (FCM). Fas monoclonal antibody was used for the blocking test. Fas expression was detected by immuocytochemistry(SABC assay) and FCM. RESULTS: VES demonstrated a significant growth inhibitory effect and apoptosis induced effect on the Tca8113 cells in a dose- and time-dependent manner. Fas neutralizing antibody can block the apoptosis induced by VES. After the administration of VES, the expression of Fas protein increased and the kytoplasm staining enhanced. Proteinum quantitative analysis showed that the mean fluorescence intensity increased. CONCLUSION: VES can induce apoptosis in human tongue cancer cells, and the up-regulation of the cell surface Fas protein may play an important role in the process.

[Relationship between the expression of cyclooxygenase-2 and microvessel density in oral squamnous cell carcinoma].

OBJECTIVE: To detective the relationship between cyclooxygenase-2 (COX-2) and angiogenesis in oral squamous cell carcinoma (OSCC) and its clinical significance through observing the expression of COX-2 and determining microvessel density (MVD) in OSCC. METHODS: PV-9000 immunohistochemistry was used to determine the expression of COX-2 and CD34, which was used to determine MVD, in 76 OSCC tissues and 12 normal oral mucosa tissues. RESULTS: Overexpression of COX-2 was detected in OSCC, and was more intense compared with normal epithelium (P < 0.001). The high expression of COX-2 in OSCC was related to neck lymphnode metastasis, tumor size, TNM stage and histological grade (P <0.05). The MVD value in COX-2-positive group was much higher than that in COX-2-negative group (P < 0.01) and that in normal oral mucosa tissues (P < 0.01). CONCLUSION: The high expression of COX-2 in OSCC was significantly associated with MVD, neck lymphnode metastasis, tumor size, TNM stage and histological grade. COX-2 might be one of the important factors in the angiogenesis of OSCC.