Isolation of 3-Hydroxy-β-ionol from Burley Tobacco

Foxtail millet (Setaria italica) proteins were fractionated into five fractions, i. e., water-, salt-, ethanol-, sodium dodecyl sulfate (SDS)- and 2-mercaptoethanol (ME)-soluble fractions, by successive extraction with various solvents from millet flour. The proportion in each fraction was 7.2, 5.6, 40, 25 and 20% respectively, of total flour nitrogen. The proteins of the ethanol- and SDS-soluble proteins were similar in amino acid composition and molecular weight distribution. More than 15 different molecular weight classes of proteins ranging from 11,000 to 150,000 were distinguished by SDS-polyacrylamide gel electrophoresis without prior reduction of their disulfide bonds. These major protein bands in the gel were estimated to be homo-olygomers (monomer, dimer, trimer, etc.) of subunit A or subunit B. The molecular weights of subunits A and B were 12,000 and 17,000, respectively. Subunits A and B were also different in amino acid composition: subunit A had higher content of methionine.     

Isolation of a New Tobacco Constituent, 5,6-Dihydro-5-hydroxy-3,6-epoxy-β-ionol,from Japanese Domestic Suifu Tobacco

Glutamine production was investigated by coupling of glutamine synthetase from Gluconobacter suboxydans with a sugar fermentation system of baker's yeast (energy generating system). Under the optimum condition, 22 mM glutamine was formed in 3 hr, and the yield was 92% based on the substrate glutamate. The first step of the process was the accumulation of fructose 1,6-diphosphate (FDP) as a reservoir of fermentation energy, in the presence of a high concentration of inorganic phosphate; and the second step was accomplished by coupling the degradation of FDP with glutamine synthetase reaction through an ADP-ATP system. The effects of enzyme concentration, additives in the reaction mixture and others on glutamine formation were investigated, and the importance of three factors was pointed out: (a) the ratio of activity of energy generating system to utilizing system, (b) contaminated enzyme(s) in the energy utilizing system and (c) the enzymatic properties of the energy utilizing system.     

N′-Isopropylnornicotine in Burley Tobacco (Nicotiana tabacum)

Allyl sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical flavor components of Allium vegetables, have been shown to inhibit benzo[a]pyrene (B[a]P)-induced carcinogenesis in animal models. As a possible mechanism of this inhibition, the effect of these volatile substances on cytochrome P450 (CYP)1 (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B[a]P was investigated using a human hepatoma cell model (HepG2). DADS and DATS inhibited the B[a]P-induced ethoxyresorufin O-deethylase (EROD) activity, a marker enzyme for CYP1, by 30-90% and 70-95% at 100-1,000 μM concentration, respectively. The cell viability, an indicator of the capacity to inhibit B[a]P bioactivation, was increased by treatments of 100-1,000 μM DADS and 10-100 μM DATS. Immunoblot results indicated that the B[a]P inducible CYP1A2 protein was suppressed by 100-1,000 μM of DADS and 10-100 μM of DATS, but CYP1A1 and 1B1 were not detectable in any microsomes. Analysis of B[a]P metabolites revealed that the level of 7,8-diol formed was significantly reduced in the DADS and DATS treated microsomes as compared to the control. The level of 9,10-diol and 4,5-diol formed was also lowered by the allyl sulfide treatments. These results suggest that the protective mechanism of allyl sulfides on B[a]P-induced carcinogenesis is possibly related with the modulation of CYP1-mediated bioactivation of B[a]P.     

4-Hydroxy-β-damascone and 4-Hydroxy-dihydro-β-damascone from Cigar Tobacco

The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomyces cerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.     

Genetic Engineering of Tobacco with Double Resistance to Both Virus and Insect

ＧｅｎｅｔｉｃＥｎｇｉｎｅｅｒｉｎｇｏｆＴｏｂａｃｃｏｗｉｔｈＤｏｕｂｌｅＲｅｓｉｓｔａｎｃｅｔｏＢｏｔｈＶｉｒｕｓａｎｄＩｎｓｅｃｔＬＩＡＮＧＸｉａｏ－ｙｏｕ;（梁晓友）ＭＩＪｉｎｇ－ｊｉｕ;（米景九）,ＰａｎＮａｉ－ｓｕｉ（潘乃隧）,ＣＨＥＮｚｈ...     

Isolation of R-(−)-3-Hydroxy-β-ionone from Burley Tobacco

The tritium-hydrogen exchange method was used to determine the total racemization of amino acid residues of four proteins (ribonuclease A, lysozyme, soybean protein and casein) during their exposure to an alkali. Tritium was incorporated with first order kinetics into these proteins during their incubation in 0.2 n NaOH at 40°C. The tritium-hydrogen exchange increased as the temperature and the alkali concentration increased. In contrast, pepsin digestibility decreased extensively in the initial stage of the treatment. This phenomenon was verified by the subsite theory of protease, that only minor racemization renders the extended range of the peptide chain around the racemized amino acids non-susceptible to pepsin. General precautions against the racemization of food protein treated with alkalis are discussed briefly in terms of the deterioration of nutrients.     

Analysis of Tobacco Headspace Volatiles Using Tenax GC or Active Carbon†

Two simple methods, the Tenax GC method and the active carbon method, have been developed to analyze the headspace volatiles of tobacco. The headspace volatiles swept by helium from cut tobacco were collected by either Tenax GC or active carbon and were analyzed by gas chromatography after desorption by heating the Tenax GC or by extracting the active carbon with dichloromethane. The gas chromatograms of the headspace volatiles of flue-cured tobacco obtained by these two methods differed. Both methods showed good reproducibility and were applied to the analysis of the headspace volatiles of American and Japanese flue-cured tobacco samples. By principal component analysis, three or four principal components were. extracted from the 15 selected peaks of the headspace volatiles of these samples that had been obtained from both the Tenax GC and the active carbon methods.     

Transgenic Tobacco with αα Mutant Gene Has Higher Tolerance to Heavy Metals

Metallothionein (MT) has two domains, α and β domain. α domain preferred to bind Cd2+and Hg2+. Mouse metallothionein mutant αα has been constructed and expressed in E.coli, which has the same stability as the nature one but has stronger affinity to heavy metals. To testify the result in vivo, αα mutant gene was cloned into plant expression vector pE3 under the CaMV 35S promoter. A transgenic tobacco was obtained by using leaf discs of tobacco (Nicotiana tabacum L. cv. NC89) to Agrobacterium-mediated ααgene transfer. Southern blotting analysis indicated that the αα mutant gene was indeed integrated into the tobacco genome; Western blot indicated that the αα mutant gene was expressed in transgenic tobacco. It was also demonstrated that the transgenic tobacco with αα mutant gene have a little higher tolerance to heavy metals than that with natural MT gene. Moreover, the transgenic tobacco can accumulate more Cd2+ in its roots than natural, so that, it can decrease the concentration of Cd2+ in its leaves.     

Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

In the present study, we focused on whether Intracellular free Ca^2＋ （[Ca^2＋],） regulates the formation of mltochondrlal permeability transition pore （MPTP） In H2O2-induced apoptosis In tobacco protoplasts. It was shown that the decrease In mltochondrlal membrane potential （△ψm） preceded the appearance of H2O2-Induced apoptosls; pretreatment with the specific MPTP Inhibitor cyclosporine A, which also Inhibits Ca^2＋ cycling by the mitochondria, effectively retarded apoptosls and the decrease In △ψm. Apoptosls and decreased △ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca^2＋ channel blocker lanthanum chloride （LaCl3） attentuated these responses. Chelation of extracellular Ca^2＋ with EGTA almost totally Inhibited apoptosls and the decrease In △ψmInduced by H2O2. The time-course of changes In [Ca^2＋]l In apoptosls was detected using the Ca^2＋ probe Fiuo-3 AM. These studies showed that [Ca^2＋]1 was Increased at the very early stage of H2O2-Induced apoptosls. The EGTA evidently Inhibited the Increase In [Ca^2＋]1 Induced by H=O=, whereas It was only partially Inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca^2＋ concentrations In tobacco protoplasts, which mainly results from the entry of extracellular Ca^2＋, to regulate mltochondrlal permeability transition. The signaling pathway of [Ca^2＋]1-medlated mltochondrlal permeability transition was associated with H2O2-Induced apoptosis In tobacco protoplaete.     

Tobacco and myocardial infarction: is snuff less dangerous than cigarettes?

OBJECTIVE--To estimate the risk of myocardial infarction in snuff users, cigarette smokers, and non-tobacco users in northern Sweden, where using snuff is traditional. DESIGN--Case-control study. SETTING--Northern Sweden. SUBJECTS--All 35-64 year old men who had had a first myocardial infarction and a population based sample of 35-64 year old men who had not had an infarction in the same geographical area. MAIN OUTCOME MEASURE--Tobacco consumption (regular snuff dipping, regular cigarette smoking, non-tobacco use) and risk of acute myocardial infarction. RESULTS--59 of 585 (10%) patients who had a first myocardial infarction and 87 of 589 (15%) randomly selected men without myocardial infarction were non-smokers who used snuff daily. The age adjusted odds ratio for myocardial infarction was 0.89 (95% confidence interval 0.62 to 1.29) for exposure to snuff and 1.87 (1.40 to 2.48) for cigarette smoking compared with non-tobacco users, showing an increased risk in smokers but not in snuff dippers. Regular cigarette smokers had a significantly higher risk of myocardial infarction than regular snuff dippers (age adjusted odds ratio 2.09; 1.39 to 3.15). Smoking, but not snuff dipping, predicted myocardial infarction in a multiple logistic regression model that included age and level of education. CONCLUSIONS--In middle aged men snuff dipping is associated with a lower risk of myocardial infarction than cigarette smoking.     

Is Salicylic Acid a Translocated Signal of Systemic Acquired Resistance in Tobacco?

Salicylic acid (SA) is a likely endogenous signal in the development of systemic acquired resistance (SAR) in some dicotyledonous plants. In tobacco mosaic virus (TMV)-resistant Xanthi-nc tobacco, SA levels increase systemically following the inoculation of a single leaf with TMV. To determine the extent to which systemic increases in SA result from SA export from the inoculated leaf, SA produced in TMV-inoculated or healthy leaves was noninvasively labeled with 18O2. Spatial and temporal distribution of 18O-SA indicated that most of the SA detected in the healthy tissues was synthesized in the inoculated leaf. No significant increase in the activity of benzoic acid 2-hydroxylase, the last enzyme involved in SA biosynthesis, was detected in upper uninoculated leaves, although the basal level of enzyme activity was relatively high. No increases in SA level, pathogenesis-related PR-1 gene expression, or TMV resistance in the upper uninoculated leaf were observed if the TMV-inoculated leaf was detached up to 60 hr after inoculation. Apart from the inoculated tissues, the highest increase in SA was observed in the leaf located directly above the inoculated leaf. The systemic SA increase observed during SAR may be explained by phloem transport of SA from the inoculation sites.     

Tobacco leaf spot and root rot caused by Rhizoctonia solani Kühn

Rhizoctonia solani Kühn is a soil-borne fungal pathogen that causes disease in a wide range of plants worldwide. Strains of the fungus are traditionally grouped into genetically isolated anastomosis groups (AGs) based on hyphal anastomosis reactions. This article summarizes aspects related to the infection process, colonization of the host and molecular mechanisms employed by tobacco plants in resistance against R. solani diseases. TAXONOMY: Teleomorph: Thanatephorus cucumeris (Frank) Donk; anamorph: Rhizoctonia solani Kühn; Kingdom Fungi; Phylum Basidiomycota; Class Agaricomycetes; Order Cantharellales; Family Ceratobasidiaceae; genus Thanatephorus. IDENTIFICATION: Somatic hyphae in culture and hyphae colonizing a substrate or host are first hyaline, then buff to dark brown in colour when aging. Hyphae tend to form at right angles at branching points that are usually constricted. Cells lack clamp connections, but possess a complex dolipore septum with continuous parenthesomes and are multinucleate. Hyphae are variable in size, ranging from 3 to 17 μm in diameter. Although the fungus does not produce any conidial structure, ellipsoid to globose, barrel-shaped cells, named monilioid cells, 10-20 μm wide, can be produced in chains and can give rise to sclerotia. Sclerotia are irregularly shaped, up to 8-10 mm in diameter and light to dark brown in colour. DISEASE SYMPTOMS: Symptoms in tobacco depend on AG as well as on the tissue being colonized. Rhizoctonia solani AG-2-2 and AG-3 infect tobacco seedlings and cause damping off and stem rot. Rhizoctonia solani AG-3 causes 'sore shin' and 'target spot' in mature tobacco plants. In general, water-soaked lesions start on leaves and extend up the stem. Stem lesions vary in colour from brown to black. During late stages, diseased leaves are easily separated from the plant because of severe wilting. In seed beds, disease areas are typically in the form of circular to irregular patches of poorly growing, yellowish and/or stunted seedlings. RESISTANCE: Knowledge is scarce regarding the mechanisms associated with resistance to R. solani in tobacco. However, recent evidence suggests a complex response that involves several constitutive factors, as well as induced barriers controlled by multiple defence pathways. MANAGEMENT: This fungus can survive for many years in soil as mycelium, and also by producing sclerotia, which makes the management of the disease using conventional means very difficult. Integrated pest management has been most successful; it includes timely fungicide applications, crop rotation and attention to soil moisture levels. Recent developments in biocontrol may provide other tools to control R. solani in tobacco.     

Isolation and Identification of γ-l-Glutamyl-l-glutamic Acid from Tobacco Cells in Suspension Culture

The Maillard Reaction (MR) rate below the glass transition temperature (T_g) for various model glassy food systems was studied at temperatures between 40 °C and 70 °C. As a sample, freeze-dried glucose and lysine systems embedded in various glassy matrices (e.g., polyvinylpyrrolodone and trehalose) were used, and the MR rate below the T_g was compared among the various glassy matrices. The extent of MR was estimated spectrophotometrically from the optical density at 280 nm (OD₂₈₀), and the MR rate (k₂₈₀) was determined as a pseudo zero order reaction rate from the time course of OD₂₈₀. Although k₂₈₀ was described by the Arrhenius plot, the temperature dependence of k₂₈₀ was almost the same and the intercept was different among the matrices. From the comparison of k₂₈₀, it was suggested that the MR rate in glassy matrix was affected not only by the T_g, but also by the hydrogen bonding between MR reactants and glassy matrix.     

Why Have Tobacco Control Policies Stalled? Using Genetic Moderation to Examine Policy Impacts

Background

Research has shown that tobacco control policies have helped produce the dramatic decline in use over the decades following the 1964 surgeon general’s report. However, prevalence rates have stagnated during the past two decades in the US, even with large tobacco taxes and expansions of clean air laws. The observed differences in tobacco control policy effectiveness and why policies do not help all smokers are largely unexplained.

Objective

The aim of this study was to determine the importance of genetics in explaining response to tobacco taxation policy by testing the potential of gene-policy interaction in determining adult tobacco use.

Methods

A moderated regression analysis framework was used to test interactive effects between genotype and tobacco policy in predicting tobacco use. Cross sectional data of US adults from the National Health and Nutrition Examination Survey (NHANES) linked with genotype and geocodes were used to identify tobacco use phenotypes, state-level taxation rates, and variation in the nicotinic acetylcholine receptor (CHRNA6) genotype. Tobacco use phenotypes included current use, number of cigarettes smoked per day, and blood serum cotinine measurements.

Results

Variation in the nicotinic acetylcholine receptor was found to moderate the influence of tobacco taxation on multiple measures of tobacco use. Individuals with the protective G/G polymorphism (51% of the sample) responded to taxation while others had no response. The estimated differences in response by genotype were C/C genotype: b = −0.016 se  = 0.018; G/C genotype: b = 0.014 se  = 0.017; G/G genotype: b = −0.071 se 0.029.

Conclusions

This study provides novel evidence of “gene-policy” interaction and suggests a genetic mechanism for the large differences in response to tobacco policies. The inability for these policies to reduce use for individuals with specific genotypes suggests alternative methods may be needed to further reduce use.     

Interaction between a Nanovirus-like Component and the Tobacco Curly Shoot Virus／Satellite Complex

The biological role of DNA1, a nanovirus-like component shown to be associated with the begomovirus/satellite complex, has not yet been identified. Here, we demonstrated that DNA1 of Tobacco curly shoot virus isolate Y35 (TbCSV-Y35) attenuated leaf-curling symptoms induced by TbCSV-Y35 or TbCSV-Y35 plus Y35 DNAβ in the early stage of symptom development and induced leaf cluster at a later stage of symptom development in Nicotiana benthamiana plants. The leaf disc assay demonstrated that TbCSV-Y35 DNA1 replicated autonomously. Southern blot analysis revealed that TbCSV-Y35 DNA1 reduced viral DNA accumulation. Viral DNA accumulation was not reduced when plants were co-inoculated with TbCSV-Y35 DNAβ, but the TbCSV-Y35 DNAβ level was dramatically reduced in the presence of TbCSV-Y35 DNA1. To determine whether the interaction between TbCSV/satellite complex and DNA1 had isolate specificity, DNA1 of TbCSV isolate Y132 was cloned and sequenced. It was found to have 75% nucleotide sequence identity with TbCSV-Y35 DNA1. Infectivity tests showed that TbCSV-Y132 DNA1 had no effect on the symptoms induced by TbCSV-Y35 or TbCSV-Y35 and Y35 DNAβ in N. benthamiana plants, although Y 132 DNA1 could replicate in these plants.     

Purification of an Apoplastic β-N-Acetyl-D-Hexosaminidase from Tomato Leaves Infected with Tobacco Mosaic Virus

Systematic infection of tomato (Lycopersicon esculenturn) leaves by tobacco mosaic virus (TMV) increased the levels of β-N-acetyl-D-hexosaminidase activity. The enzyme was purified from intercellular fluid by --20℃ acetone precipitation, CM-Sephadex C-25 ion exchange chromatography, Polybuffer Exchanger 94 chromatofocusing and Sephadex G-150 gel filtration column to homogeneity. The molecular weight obtained by SDS-PAGE and Sephadex G-150 gel filtration was 75 kD and 145 kD respectively. The enzyme hydrolysed p-nitrophenyl-N-acetyl-β-glucosaminide and p-nitrophenyl-N-acetyl-β-galaetosaminide, it was a glycoprotein. Most of the enzyme activity in the TMV-infected tomato leaves was found in the intercellular spaces.     

Transport of Cl– across the Plasma Membrane during Pollen Grain Germination in Tobacco

The role of Cl- transport across the plasma membrane was studied in an early step of pollen grain germination in tobacco Nicotiana tabacum L. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid, completely suppress the germination with IC(50) approximately 8 micro M. At this concentration NPPB reduces the rate of Cl- efflux out of pollen grain by 1.8-fold in the interval 5-12 min, and niflumic acid reduces the rate 1.2-fold. 4,4;-Diisothiocyanatostilbene-2,2;-disulfonic acid, a known inhibitor of Cl- channels and antiporters, completely suppresses germination as well (IC(50) = 240 micro M), but has no effect on the rate of Cl- efflux. Inhibitors of chloride co-transporters, such as furosemide, bumetanide, and bis(1,3-dibutylbarbituric acid)pentamethine oxonol, suppress the germination by less than 50%. This set of data suggests that NPPB-sensitive anion channels are involved in the activation of pollen grains in the early stage of germination.