Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro

The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca^2＋ and 8 mM Mg^2＋. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 μm in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.

Torin 1,TOR Inhibitor Enhances Cellular Proliferation in NT-1 Tobacco Suspension Cell Cultures

Torin 1 is an ATP-competitive TOR inhibitor which inhibits the signaling of TOR and S6K kinase in mammals and plants.The objective of this research is to determine the effect of Torin 1 in a relatively simple and homogeneous plant system such as the NT-1 tobacco suspension cell cultures.Cultures of NT-1 cells were tested with 5,50,150 and 250 nM of Torin 1.During kinetics growth of NT-1 tobacco suspension cell cultures,150 and 250 nM Torin 1 inhibits the early growth and later enhanced the cellular proliferation during exponential growth by means of an increased expression of E2F1 and cyclin B.Furthermore,Torin 1 stimulates the growth of NT-1 cells during log phase with small shaped cell,characteristic of tobacco suspension cell cultures with high mitotic activity.

Alcohol Dehydrogenase Activity in Cultured Cells from Different Tobacco (Nicotiana tabacum L.) Varieties

The activity of alcohol dehydrogenase (ADH) in cultured cells of various tobacco was determined. It was found that significant differences existed in cells of different varieties cultured under normal conditions and as well after treated with exogenous ethanol. The ADH activity had positive relation with the ability of the cells to catabolize exogenous ethanol, indicating that the main function of the ADH in tobacco cells was in the direction of converting ethanol to acetaldehyde.

Gene Expression Changes and Signal Transduction Pathway Alterations in Primary Human Oral Epithelial Cells Exposed to Smokeless Tobacco Extracts

Smokeless tobacco (ST), an alternative to smoking, has gained wide popularity among tobacco users. This study is conducted to determine the time course of gene expression associated with specific signaling pathways in human oral epithelial cells after exposure to smokeless tobacco extract (STE). A differentiated layer of epithelial cell is created as a way to mimic reasonably similar physiological atmosphere. A dose and time dependent response is observed for cell viability and cell proliferation assays indicating that this model system is responsive to the treatment. Expressions of 84 genes representing 18 different signal transduction pathways are quantitated. This is accomplished by using real-time polymerase chain reaction arrays at 1 h, 3 h, 6 h and 24 h time points following exposure to STE. Changes in gene expression are observed on many cellular processes including cell cycle regulation, cell adhesion, inflammation, apoptosis, and DNA breaks-down including Akt pathway activation. Short time exposure (1 h) leads more genes to down regulate whereas longer incubation time results in more genes up regulation. Most notable differences in the expression of genes during the course of treatment are BCL2A1, BIRC3, CCL20, CDK2, EGR1, FOXA2, HOXA1, IGFBP3, IL1A, IL-8, MMP10, NOS2, NRIP1, PTGS2, SELPLG and TNF-a. This study provides an insight on gene expression on oral epithelial cells as a result of STE exposure. This may also postulate greater understanding on biological effects and the mechanism of action of STE particularly at the transcriptional level.

Atrophic gastritis: Risk factor for esophageal squamous cell carcinoma in a Latin-American population

AIM: To study the association between atrophic gastritis (AG) and esophageal squamous cell carcinoma (ESCC) in a Latin-America population. METHODS: A case-control study was performed at two reference Brazilian hospitals including patients diagnosed with advanced ESCC and dyspeptic patients who had been subjected to upper gastrointestinal endoscopy, with biopsies of the gastric antrum and body.All cases with ESCC were reviewed by a single pathologist, who applied standard criteria for the diagnosis of mucosal atrophy, intestinal metaplasia, and dysplasia, all classified as AG. The data on the patients' age, sex, smoking status, and alcohol consumption were collected from clinical records, and any missing information was completed by telephone interview. The association between AG and ESCC was assessed by means of univariate and multiple conditional logistic regressions. RESULTS: Most patients were male, and the median age was 59 years (range: 37-79 years) in both the ESCC and control groups. Univariate analysis showed that an intake of ethanol greater than 32 g/d was an independent risk factor that increased the odds of ESCC 7.57 times (P = 0.014); upon multiple analysis, alcohol intake of ethanol greater than 32 g/d exhibited a risk of 4.54 (P = 0.081), as adjusted for AG and smoking. Smoking was shown to be an independent risk factor that increased the odds of ESCC 14.55 times (P = 0.011) for individuals who smoked 0 to 51 packs/year and 21.40 times (P = 0.006) for those who smoked more than 51 packs/year. Upon multiple analyses, those who smoked up to 51 packs/year exhibited a risk of 7.85 (P = 0.058), and those who smoked more than 51 packs/ year had a risk 11.57 times higher (P = 0.04), as adjusted for AG and alcohol consumption. AG proved to be a risk factor that increased the odds of ESCC 5.33 times (95%CI: 1.55-18.30, P = 0.008) according to the results of univariate conditional logistic regression. CONCLUSION: There was an association by univariate conditional logistic regression between AG and ECSS in this sample of Latin-American population.

Screening of Total Organophosphate Pesticides in Agricultural Products with a Cellular Biosensor

Organophosphates belong to the most important pesticides used in agricultural practice worldwide. Although their analytical determinations are quite feasible with various conventional methods, there is a lack of efficient screening methods, which will facilitate the rapid, high-throughput detection of organophosphates in different food commodities. This study presents the construction of a rapid and sensitive cellular biosensor test based on the measurement of changes of the cell membrane potential of immobilized cells, according to the working principle of the Bioelectric Recognition Assay (BERA). Two different cell types were used, derived either by animal (neuroblastoma) or plant cells (tobacco protoplasts). The sensor was applied for the detection of a mixture of two organophosphate pesticides, diazinon and chlorpyrifos in two different substrates (tomato, orange). The pesticides in the samples inhibited the activity of cell membrane-bound acetylcholinesterase (AChE), thus causing a measurable membrane depolarization in the presence of achetylcholine (Ach). Based on the observed patterns of response, we demonstrate that the sensor can be used for the qualitative and, in some concentrations, quantitative detection of organophosphates in different substrates with satisfactory reproducibility and sensitivity, with a limit of detection at least equal to the official Limit of Detection (LOQ). The assay is rapid with a total duration of 3 min at a competitive cost. The sensitivity of the biosensor can be further increased either by incorporating more AChE-bearing cells per test reaction unit or by using cells engineered with more potent AChE isoforms. Standardization of cultured cell parameters, such as age of the cells and subculture history prior to cell immobilization, combined with use of planar electrodes, can further increase the reproducibility of the novel test.

Relationship Between LOX Activity, SA and JA Accumulation in Tobacco Leaves Under Water Stress

The relationship between LOX(lipoxygenase)activity, SA(salicylic acid)and JA(jasmonic acid)accumulation was studied in the tobacco leaf under water stress. The result showed that LOX activity and JA content increased simutaneously with losing of water . NDGA(nordihydroguaiaretic),an inhibitor of LOX, inhibited simultaneously both the activity of LOX and the enhancement of JA level under the stress. Likewise, adding LOX to tobacco cell-free system led to the increase of JA content. It suggested that LOX could be a key enzyme in JA biosynthesis under water stress. SA inhibited the enhancement of JA level under the stress.

Effects of Obesity and Smoking on Survival in Non-Small Cell Lung Cancer

Background: Obesity is an emerging leading cause of morbidity and mortality in the US and the relationship between obesity, tobacco, and survival in NSCLC is unclear. Methods: Data (n = 87,631) were obtained from linkage of the 1996-2007 Florida Cancer Data System to the Agency for Health Care Administration database providing procedure and diagnoses codes. Survival time was calculated from date of diagnosis to date of death. Smoking status was categorized as never, current, and former. Obesity (yes/no) = ICD9 code BMI > 30 kg/m2, cachexia = ICD9 code “wasting syndrome”, & non-obese = non-obese & non cachectic. Cox proportional regression models used to predict survival;demographic, clinical, treatment factors, & comorbidities were included in adjusted models with smoking status and obesity as the main factors. Results: The majority of patients (pts) were either former (49%) or current (40%) smokers, & non-obese (88%). 6.8% of pts were obese & 4.8% of pts were cachectic. There were significant differences between survival curves and median survival (months) for obese vs. non-obese vs. cachectic pts. (20 vs 10 vs. 7.9;P < 0.001). Former and current smokers had shorter median survival than never smokers (10.8 & 9.2 vs. 11.9;P < 0.001). Survival rates (%) at 1-yr (60.1 vs. 45.2 vs. 37.7;P < 0.001), 5-yr (30.3 vs. 15.4 vs. 9.5;P < 0.001), 10-yr (18.1 vs. 7.6 vs. 2.7;P < 0.001) were better for obese vs. non-obese and cachectic pts respectively. Independent predictor of worse survival in the unadjusted model was former (HR 1.08;P < 0.001) and current (HR 1.20;P < 0.001) smokers compared to never. Obese and non-obese pts had better survival vs. cachexia pts. (HR 0.52;P < 0.001 and HR 0.80, p < 0.001 respectively) and obese had better survival than Non-obese pts (HR 0.65, p < 0.001). In the adjusted model, controlling for extensive variables and comorbidities, former (HR 1.11;P < 0.001) and current (HR 1.19;P < 0.001) smokers still had significantly worse survival vs. never smokers. Obese patients still had better survival (HR 0.87;P < 0.001, and HR 0.88, p < 0.001) vs. cachexia patients and non-obese respectively, survival rate was not significantly different compare non-obese with cachexia. Conclusions: Our results show that being a former or current smoker worsens survival while obesity improved survival when compared with cachexia patients or Non-obese.

Effects of environmental stressors on stem cells

Environmental toxicants are ubiquitous,and many are known to cause harmful health effects.However,much of what we know or think we know concerning the targets and long-term effects of exposure to environmental stressors is sadly lacking.Toxicant exposure may have health effects that are currently mischaracterized or at least mechanistically incompletely understood.While much of the recent excitement about stem cells(SCs)focuses on their potential as therapeutic agents,they also offer a valuable resource to give us insight into the mechanisms and risks of toxicant effects.Not only as a response to the increasing ethical pressure to reduce animal testing,SC studies allow us valuable insight into the true effects of human exposure to environmental stressors under controlled conditions.We present a review of the history of publications on the effects of environmental stressors on SCs,followed by a consolidation of the literature over the past five years on a subset of key environmental stressors of importance to human health and their effects on both embryonic and tissue SCs.The review will make constructive suggestions as to areas of toxicant research where further studies are needed,as well as making indications of the potential utility for advancing knowledge and directing research on environmental toxicology.

不同基因型烤烟成熟期细胞壁物质及相关酶活性变化

为了解不同烟叶成熟度细胞壁物质含量及其酶活性的动态变化规律,为烟叶适时采收、烟叶品质提升提供一定理论依据,以烤烟品种NC71、中烟100和NX211的中部烟叶为材料,研究不同烤烟品种在成熟衰老过程中细胞壁组分含量及其相关酶活性动态变化。结果表明,随着叶片成熟与衰老进程的不断推进,总果胶和纤维素含量均在适熟时达到最高,其中,总果胶含量大小表现为NC71>中烟100>NX211,但差异不显著,纤维素含量大小表现为中烟100>NC71>NX211,但差异不显著;木质素含量从欠熟到适熟呈上升的趋势;果胶酯酶(PE)在适熟时活性最低,其大小表现为NC71>中烟100>NX211,但NC71和中烟100差异不显著;多聚半乳糖醛酸酶(PG)在适熟时活性最高,其大小表现为中烟100>NX211>NC71,各品种间PG活性差异显著;纤维素酶在完熟时活性达到最高,其大小表现为中烟100>NC71>NX211,但中烟100和NC71差异不显著;不同烤烟品种细胞壁组分含量及相关酶活性变化趋势相同,但在量和活性值上有所差异。根据不同烤烟品种在成熟期细胞壁物质的变化规律,可有针对性地选择适宜采收期。

Floral gradient in flowering tobacco in relation to free amino acids

By employing TCLs (thin cell layers) culture, the floral gradient in flowering tobacco of different developmental stages was confirmed. The TCLs from early flowering tobacco regenerated more floral buds than those from the tobacco plants in full blooming or fruiting stages. Analysis of free amino acid levels revealed the acropetal gradient of Pro in flowering tobacco stem. L-Pro. L-Trp. D,L-Met and L-Arg were respectively added into the culture medium for testing their influence on floral bud formation from tobacco pedicel segments. Only L-Trp evidently enhanced the floral bud neoformation.

免疫启动前烟草烟雾暴露促进EAE发病的细胞免疫机制初步研究

目的探讨自身免疫激活前短暂烟草烟雾暴露对成年SD大鼠实验性自身免疫性脑脊髓炎(EAE)发病及脊髓炎症反应的影响。方法采用随机数字表法将30只SD大鼠分为空白对照组、空气组和烟草组。在EAE造模前1周予以连续烟草烟雾暴露7 d,观察各组动物神经行为学变化并评分。在EAE造模后第21天抽取脑脊液并处死动物,采用ELISA法检测脑脊液IL-17F、IL-10水平,流式细胞术检测脾脏CD4^(+)T细胞亚型变化。颈髓、胸髓、腰髓石蜡切片经HE染色、监牢蓝髓鞘染色后评估炎症反应及脱髓鞘百分比。结果烟草组最高行为学评分高于空气组[3.50(1.00,4.00)分比2.00(0,2.25)分],差异有统计学意义(P<0.05)。烟草组颈髓、胸髓、腰髓HE染色病理评分和脱髓鞘百分比均高于空气组,差异均有统计学意义(均P<0.05)。烟草组抗炎的CD4^(+)叉头蛋白P3(FOXP3)^(+)T细胞百分比低于空气组,而具有致病性的CD4^(+)FOXP3^(-)T细胞百分比高于空气组,差异均有统计学意义(均P<0.05)。烟草组脑脊液IL-17F水平高于空气组,IL-10水平低于空气组,差异均有统计学意义(均P<0.05)。结论免疫激活前烟草烟雾暴露可加重成年大鼠EAE神经功能受损,并促进脊髓炎症反应。

超表达CsCslE1_4基因对烟草细胞壁成分的影响

类纤维素合酶E(CslE)基因与纤维素合成相关,决定着细胞形状以及大小,并在植物生长发育阶段起着机械支撑和抵抗病原物侵染的作用。为研究CsCslE1_4基因对植物细胞壁的重要组成成分的影响,应用转基因烟草研究CsCslE1_4基因的基本功能,通过农杆菌介导法遗传转化烟草,经鉴定得到18株阳性植株,结合定量表达以及细胞壁组成成分测定。结果表明,与野生型相比,转基因株系茎的纤维素含量是野生型的1.43~5.48倍,叶中纤维素含量除株系CslE2外,其余株系纤维素含量是野生型的1.08~2.49倍,大部分转基因株系半纤维素含量是野生型半纤维素含量的1.18~2.22倍,但果胶含量变化趋势与纤维素和半纤维素含量变化趋势相反,木质素含量没有明显的变化。CsCslE1_4基因可以促进纤维素合成,这为后续解析基因功能以及抗逆性提供依据。

云南晾晒烟中小分子的荧光光谱及显微成像

为提高晾晒烟中化学成分的研究利用,进一步拓展烟草资源的用途,以云南地方晾晒烟为研究对象,分离得到2个荧光小分子化合物2-甲基喹啉(QND)和3-羧酸吲哚(YD),对其进行了光谱分析和药理学毒性与成像分析. 2个荧光小分子均显示出较高的荧光量子产率,在一定范围的p H条件下,对环境p H的变化表现出良好的响应,且呈现互补特征.细胞实验结果表明,实验条件下两个小分子细胞毒性较低,细胞成像结果均呈现出较好的荧光成像能力,具有在生物领域应用的可行性,为晾晒烟资源的综合应用以及探究烟草的新用途新用法展示出良好的应用前景.

男性烟依赖者CDH23基因甲基化与颈动脉粥样硬化关系

目的探讨男性烟依赖者CDH23基因位点甲基化改变及其与颈动脉粥样硬化发生的关系。方法选取青岛某三甲医院健康查体男性130例作为研究对象,有烟依赖史者64例纳入烟依赖组,无烟依赖史者66例纳入对照组。应用问卷调查方式搜集两组研究对象一般资料,包括年龄、受教育程度、既往疾病史、烟依赖史等;采用彩色多普勒超声仪器检测颈动脉内膜厚度;采集血样标本,检测CDH23基因位点甲基化数值;分析男性烟依赖者CDH23基因位点甲基化改变及其与颈动脉粥样硬化的关系。结果烟依赖组和对照组一般资料包括年龄、文化程度、既往疾病史比较,差异无统计学意义(P>0.05);烟依赖组CpG246、CpG48两个位点的甲基化程度低于对照组,差异有统计学意义(t=-2.454、-2.436,P<0.05);CpG246、CpG48位点甲基化水平与颈动脉内膜厚度相关(F=6.235、4.692,P<0.05),低甲基化水平时颈动脉内膜厚度显著大于高甲基化水平。结论烟依赖者CDH23基因CpG位点发生低甲基化改变,这种低甲基化改变与颈动脉粥样硬化发生密切相关。

烟草内生真菌Penicillium janthinellum的代谢表型分析

为了研究烟草内生真菌Penicillium janthinellum的代谢表型特征,本文利用Biolog细胞表型芯片技术,对烟草内生真菌的950种代谢表型进行了测定。结果表明:烟草内生真菌能够代谢101种碳源、304种氮源、8种磷源和1种硫源;能在10%氯化钠、6%氯化钾、5%硫酸钠、20%乙二醇、6%甲酸钠、7%尿素、12%乳酸钠、200 mmol/L磷酸钠、100 mmol/L硝酸钠、100 mmol/L硫酸铵中较好生长;其适应的pH值范围为5~10;当pH值为5.2时,在20~200 mmol/L苯甲酸钠中无法生存。

密集烘烤过程中烤烟细胞生理和质地变化

采用河南农业大学设计的电热式温湿自控密集烤烟箱,以中烟100上部叶为材料,研究了烘烤过程中烟叶细胞生理和质地变化的规律。结果表明,烘烤中烟叶的呼吸强度在38℃时达到高峰,箱内CO2含量为1.80%,48℃以后烟叶的呼吸作用减弱。烘烤中果胶酯酶和纤维素酶在38~54℃活性较高,其中果胶甲酯酶（PME）和纤维素酶活性42℃最高,多聚半乳糖醛酸酶（PG）活性48℃最高,PG与PME共同促进了原果胶的水解进程,烘烤中原果胶、总果胶和纤维素含量不断降低。烟叶质地（除黏聚性）在38~48℃之间发生显著变化,其中42℃硬度和咀嚼性值最大,38℃拉力和剪切力值最大,48℃黏聚性、拉力和剪切力值最小。相关分析表明,可溶性果胶与回复性呈显著负相关（P〈0.05）;原果胶、总果胶及纤维素与回复性、拉力均呈显著或极显著正相关（P〈0.05）。这些结果说明烘烤过程中烟叶质地的绵软在某种程度上是细胞壁物质不断降解的结果。研究表明,烘烤中烟叶剪切力的动态变化较为直观,回复性和拉力可以作为探讨烟叶细胞生理变化的参数,而烟叶细胞生理变化是导致质地差异显著的主要因素。

流式细胞术分析强声波对植物细胞周期的影响

应用流式细胞术分析烟草细胞在交变应力作用下细胞周期的变化。用特制的强声波发生装置产生频率和强度可调的交变应力场 ,研究不同频率和强度的交变应力作用后烟草细胞周期的变化。实验结果表明 ,在交变应力作用下直接影响细胞或细胞分裂的同步化 ,促进S期的DNA合成 ,有助于细胞有丝分裂。声波频率在400Hz至800Hz,强度在90dB到110dB内 ,随频率和强度的增加 ,交变应力使S期细胞明显增加 ,但频率或强度过大 ,反而使S期细胞大大减少。

施氮量对烤烟成熟期中部烟叶碳氮代谢及相关基因表达的影响

为探明不同施氮量处理对烤烟碳氮代谢的影响,以烤烟品种豫烟10号为材料进行试验,施氮量分别设置低氮(22.5 kg/hm^2)、中氮(26.25 kg/hm^2)和高氮(30 kg/hm^2)3个处理,研究烤烟成熟期中部烟叶细胞的超微结构、碳氮代谢产物的变化规律和碳氮代谢途径相关基因的表达模式。结果表明:随土壤氮肥施用量的增加,烟叶细胞中淀粉粒体积增大,烟叶总糖和还原糖含量降低,淀粉和总氮含量增加。碳代谢关键基因AGPase的表达量随烟叶的生长而增加,但增加量随施氮量的增加而逐渐减少;氮代谢关键基因NR的表达量则随施氮量的增加而下降。

烤烟细胞壁物质对烟叶质量影响研究

为了解烤烟细胞壁物质与烟叶质量的相关,测定了"金攀西"优质烟开发区攀枝花和凉山州烤烟B2F、C3F、X2F三个等级烟叶细胞壁成分;并对四川烤烟细胞壁成分和烟叶质量的数据进行偏相关分析。结果表明:(1)四川烤烟B2F、C3F、X2F三个等级间烟叶细胞壁成分含量差异较小,烟叶的总细胞壁物质、全纤维素、木质素、果胶、半纤维素、a纤维素含量分别集中在24%～35%、10%～16%、0.5%～4%、5%～9%、2%～5%、5%～9%之间。(2)四川烤烟烟叶总细胞壁物质含量与身份、抗张力、香气质得分显著负相关,与平衡含水率显著正相关。烟叶全纤维素含量与身份、厚度显著正相关。烟叶半纤维素含量与身份显著正相关。烟叶a纤维素含量与结构显著正相关,与身份显著负相关。