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.

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。

应用圆二色光谱研究交变应力对烟草细胞膜蛋白结构的影响

本文用圆二色光谱 (CD)研究了交变应力对烟草细胞膜蛋白结构的影响 采用本实验室研制的强声波发生装置来产生交变应力场 ,研究了不同强度和频率的交变应力场作用后烟草细胞膜蛋白结构的变化 研究结果表明 :细胞膜蛋白结构变化与应力的频率和强度密切相关 ,一定频率和强度范围内的交变应力能使得烟草细胞膜蛋白的二级结构发生明显的变化 ,表现为α螺旋的增加和 β转角的减少 这反映了细胞膜蛋白和磷脂相互作用的增强和细胞膜流动性的增加 ,进而为细胞的生长和分裂提供有利的条件 从细胞和分子水平研究交变应力对植物生长。

烤烟细胞壁物质与烟叶质量的关系研究

烟草细胞壁物质由非活性物质组成,为烟草提供结构框架的物质。研究了烤烟叶片细胞壁物质的组成和含量,分析了烤烟叶片细胞壁物质与烟叶等级、部位、颜色、身份、油分、物理特性、化学成分、评吸质量的关系及其调控措施。

四川烤烟烟叶细胞壁物质含量与外观质量、感官质量的关系

为了探讨烤烟细胞壁物质含量与烟叶质量的关系,对四川烤烟烟叶细胞壁物质含量进行了测定,并分析了其与外观质量、感官质量的关系。结果表明,四川烤烟烟叶中总细胞壁物质、全纤维素、木质素、果胶、半纤维素、纤维素含量范围分别为24%～35%,10%～16%,0.5%～4%,5%～9%,2%～5%,5%～9%。总细胞壁物质含量、全纤维素含量、半纤维素含量、纤维素含量与身份(外观质量)呈显著相关关系,与其他外观质量指标相关性不显著。总细胞壁物质含量与香气质(感官质量)得分显著负相关,与其他感官质量指标相关性不显著。

干旱对上部烟叶细胞结构和化学成分的影响

研究了烟草在土壤含水量为 1 2 %、1 6%、2 0 %的条件下叶片栅栏组织细胞的结构和烟草化学成分的变化规律 .结果表明 :干旱处理的烟草 ,叶片栅栏组织细胞内淀粉粒变小 ,数量变少 .叶片中还原性糖、可溶性总糖和 K2 O含量增加 ,淀粉和烟碱含量减少 .干旱程度越深 ,其变化趋势越明显 .

硅对盐胁迫烟草悬浮细胞的影响

研究了硅对盐胁迫烟草 (NicotianatabacumL .)悬浮细胞的影响。结果表明 ,硅通过提高盐胁迫细胞胞外过氧化物酶POD活性、改善细胞的长宽比例使细胞更趋向于圆形和减少盐胁迫引起的畸形细胞等作用使细胞保持较旺盛的分裂和代谢能力 ,硅可进一步调节细胞内水分状况 ,增强细胞的耐盐性。本文对这一影响的可能机制进行了讨论。

典型烤烟产区初烤烟叶表面细胞形态特征分析

为了解我国烤烟表面细胞形态特征及差异,选择典型产区相同等级(C3F)的初烤烟叶,采用扫描电镜获取烟叶表面微观图像,利用软件量化分析了细胞面积、周长、形状因子等指标,初步探讨了其与外观品质的关系。结果表明:①不同产区烟叶表面细胞形态特征差异明显并呈一定的区域变化规律,广东和福建采样点烟叶的细胞面积明显较大,细胞形状相对不规则;辽宁和河南采样点烟叶细胞形状相对规则,但辽宁采样点烟叶细胞面积明显较小,细胞褶皱感(凹凸感)较强,而河南采样点烟叶细胞面积相对偏大,褶皱感较弱;云南和贵州采样点烟叶细胞面积相对中等,贵州采样点烟叶细胞形状相对不规则;四川和湖北采样点烟叶细胞面积相对偏小,细胞形状规则程度中等。②细胞面积和周长与叶片结构和油分分值呈显著或极显著负相关,细胞形状规则程度与身份分值呈极显著负相关。

酶解细胞壁对低等级烟叶品质的改良作用

【目的】考察多糖复合酶、中性蛋白酶、果胶酶、淀粉酶对低等级烟叶细胞壁物质的降解效果,以提高低等级烟叶的品质和利用价值。【方法】以亳州X3L下部烟叶为研究对象,经多糖复合酶、中性蛋白酶、果胶酶、淀粉酶处理后,通过感官评定试验及细胞壁物质含量变化确定对感官品质改良效果最优的酶类;结合实际生产工艺和正交试验得到复配酶解低等级烟叶的工艺条件;并通过扫描电镜试验、热失重试验及GC-MS测定烟叶中中性致香成分含量的变化,考察酶解处理对烟叶性质的影响。【结果】多糖复合酶和中性蛋白酶对低等级烟叶细胞壁物质降解效果适中;复配酶解低等级烟叶的最佳工艺条件为:多糖复合酶用量490U/g,蛋白酶用量208U/g,酶解时间4h;经过酶处理后的烟叶表面结构发生了一定程度的变化,表面褶皱减少;酶处理对烟叶燃烧性质没有较大影响;酶处理提高了低等级烟叶中的中性致香成分含量。【结论】多糖复合酶与蛋白酶复配处理提高了低等级烟叶的品质,其处理工艺可应用于工业生产。