Cell death-mediated antiviral effect of chitosan in tobacco.

The antiviral activity induced by chitosan (CHT), and the mechanisms underlying it, were studied in a tobacco-tobacco necrosis necrovirus (TNV) pathosystem. Treatments with 0.1% CHT enhanced tobacco inducible defenses against TNV, reducing significantly the virus-induced necrotic lesions (in a range from 32% to 83%). In planta, this resistance was associated with a network of callose deposits, micro-oxidative bursts and micro-hypersensitive responses (micro-HRs), as assessed, respectively, by aniline blue, 3,3'-diaminobenzidine (DAB) and Evans blue staining. In order to verify if CHT-elicited cell death could be regarded as an apoptotic process, tobacco bright yellow 2 (BY2) cell cultures were treated with different CHT concentrations, ranging from 0.01% to 0.1%. After 6 h about half of the cultured cells incubated in 0.05% CHT were Evans blue positive, showing some typical morphological features of apoptosis, such as cytoplasm shrinkage and nuclear chromatin condensation. The latter was checked by 4',6-diamino-2-phenylindole (DAPI) and ethidium bromide nuclear staining and was visible already at 2 h after treatment. Moreover, the cell death kinetic induced by CHT was delayed by Verapamil(R), a calcium channel blocker. Finally, electrophoresis of genomic DNA extracted from cultured cell after 48 h treatment showed internucleosomal fragmentation, visualized as a distinct ladder of DNA bands corresponding to oligonucleosomal units.

Ds-Induced Alleles at the OPAQUE-2 Locus of Maize.

Transposon mutagenesis has been used to isolate mutable alleles at the Opaque-2 (O2) locus of maize. Plants with the Activator-Dissociation (Ac-Ds) system of transposable elements and O2 were crossed as males to a stable o2 tester line. Among a population of 200,000 kernels, 198 exceptional kernels with somatic instability were recovered. In four cases, designated O2-m1, o2-m2, O2-m3 and O2-m4, variegated phenotypes appeared in F(2) and subsequent generations. Genetic analyses indicated that the presence of Ds near or within the O2 gene was responsible for the observed somatic instability at the O2 locus. The phenotypes of the newly induced alleles were of two types. Alleles O2-m1, O2-m3 and O2-m4, in the absence of Ac, were characterized by kernel phenotypes indistinguishable from the wild type; in the presence of Ac they generated kernels with opaque sectors interspersed within a vitreous background. In contrast, the mutable allele o2-m2, in the absence of Ac, was characterized by kernels with a recessive phenotype similar to o2 recessive mutants. In the presence of Ac, it reverted somatically to wild-type-producing kernels with vitreous spots in an o2 background. The association of the Ds element with the O2 locus may prove a valuable tool directed to the isolation of DNA fragments bearing the O2 gene.

Clustering of genes for 20 kd zein subunits in the short arm of maize chromosome 7.

Zein is the major storage protein of the endosperm of maize kernels. When this alcohol-soluble protein is subjected to SDS polyacrylamide gel electrophoresis, it is resolved into four fractions of different molecular weight: 10, 14, 20 and 22 kilodaltons (kd). Each fraction is heterogeneous with respect to isoelectric pH. For example, the 20 kd fraction contains at least seven subfractions as revealed by isoelectric focusing in polyacrylamide gels. In this report, we present evidence that the structural genes coding for the 20 kd proteins are clustered on the short arm of chromosome 7, a region that also bears loci regulating endosperm zein biosynthesis [opaque-2 (02) and defective endosperm-B30 (De(*)-B30)]. The organization of these zein genes suggests that the evolution of at least some of the maize genome has occurred as the result of repeated duplication and divergence of chromosome segments.

A Mutant of Arabidopsis thaliana with a Reduced Response to Fusicoccin. I.

Because fusicoccin (FC) has the the capacity to promote solute uptake, a selective procedure for isolating mutants of Arabidopsis thaliana with a reduced response to the toxin has been developed. The procedure is based on the incubation of A. thaliana seedlings in a solution containing the cation Paraquat (Pq) at a concentration that per se does not produce bleaching of the leaves upon illumination but does in the presence of FC because of the increased uptake of the toxic cation. Using this procedure, we identified, among the progenies of 2010 M1 ethyl methanesulfonate-mutagenized plants, two mutants that stay green after exposure to FC and Pq. Some properties and inheritance of one of the two mutants (5-2) are described. Morphology of mutant plants is almost indistinguishable from that of the wild type. However, 5-2 seeds germinate and produce viable seedlings in the presence of FC plus the aminoglycoside antibiotic hygromycin B: plants of the mutant do not wilt when exposed to FC and stomata do not open or open only partially. In the presence of FC, the mutant appears less responsive than the wild type as far as the increment in fresh weight, the enlargement of leaf disc area, or the stimulation of H+ extrusion is concerned. Inheritance of the trait is monogenic dominant or semidominant, depending on the test used.

Complementation of a yeast delta pkc1 mutant by the Arabidopsis proteinANT.

The Saccharomyces cerevisiae protein kinase C homologue, PKC1, is involved in maintenance of cell integrity during polarized growth. We have used a mutant complementation approach to investigate related signal transduction pathways in higher plants. Here we report the isolation of a cDNA from Arabidopsis thaliana which partially suppresses the lytic defect of a delta pkc1 yeast strain. The encoded protein, ANT, belongs to the AP2-related gene family and is essential for ovule development. Expression in yeast of a LexA-ANT fusion protein activates transcription of a reporter gene from promoters containing lexA operators. Our results support the idea that ANT acts as transcriptional activator in planta.

Preparation and functional characterization of thylakoids from Arabidopsis thaliana.

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O(2) and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate.

A non-destructive selection method for resistance to fusicoccin in Arabidopsis thaliana.

A mutagenised population of seeds of Arabidopsis thaliana was allowed to germinate in the presence of the positively charged aminoglycoside hygromycin (4 µg/ml) and the fungal toxin fusicoccin (5×10-6 M). This hygromycin concentration, which is non-toxic by itself, becomes toxic when used together with fusicoccin, which stimulates cation uptake. Seeds that had germinated after 3-5 days and produced seedlings with green cotyledons were potentially resistant to fusicoccin and were therefore transferred into sterile Magenta vessels containing 1/2-strength Murashige and Skoog medium. This selection procedure is non-destructive, i.e. it allows the recovery of viable seedlings and their growth into adult plants thus permitting direct physiological characterisation.

Reference values of urinary ethylenethiourea in four regions of Italy (multicentric study).

The results of a study in which urinary ethylenethiourea (ETU) was assayed in the general population (167 subjects) of four Regions of Italy (Veneto, Lombardy, Piedmont and Trentino Alto Adige) are reported. The results are compared with those in a population of 97 subjects from Rovescala, a hillside wine-producing town a few kilometers from Pavia, where ethylenebisdithiocarbamates are sprayed by helicopter. It was found that an average of 24% of the populations of the four regions, taken together, had urinary ETU levels above detection limits (1.0 microgram 1(-1)) as compared to 37% of the population of Rovescala. The ranges of concentration were 0.8-8.3 micrograms 1(-1) for the four regions and 0.9-61.4 micrograms 1(-1) for Rovescala. Statistically significant variables for urinary ETU levels were smoking and wine drinking.

[Solvent pollution in shoe factories]. / L'inquinamento da solventi nei calzaturifici.

In order to study solvent exposure in shoe factories, 43 kinds of glues and 22 solvent products used in footwear manufacturing were analyzed. A gas chromatographic spectrometric method was used to identify the mixtures of solvents contained in glues and their diluents. Acetone, ethylacetate and cyclohexane were the solvents more frequently found in glues. Cyclohexane represented on average about 40% of the solvent mixture. Methyl ethyl ketone, 3-methylpentane and 2-methylpentane were often present in glues (45-52% of the samples), but only in a few cases were they associated with n-hexane. N-hexane and methylcyclopentane were found in 32% of the glue samples. N-hexane represented 47% of the solvents only in one glue. Most of the glues contained less than 10% n-hexane. Other solvents (dichloropropane, toluene, trichloroethane, butyl acetate, iso-butyl acetate and 2,2-dimethylbutane) were found in few glue samples or in low percentages. The 22 solvents used as glue diluents were mainly acetone, ethylacetate, dichloromethane and methyl ethyl ketone. The results suggest that solvent exposure in shoe factories has changed compared with data reported about 10 years ago. Biological monitoring of shoe factory workers should measure exposures to the specific solvents found in each factory, especially acetone, cyclohexane, ethylacetate and methyl ethyl ketone.

[Urban air pollutant exposure among traffic policemen]. / Esposizione agli inquinanti dell'aria urbana dei vigili municipali.

Exposure to dusts and benzene was studied in 65 traffic policemen. Samples of total dusts showed that mean personal exposure was 0.44 (SD = 0.30) mg/m3, with peaks of about 2 mg/m3. Exposure to 1-nitropyrene (1-NP), the main compound occurring in emissions from diesel engines, which was estimated from concentrations in dusts collected with high-flow samplers, was 0.28 (SD = 0.19) ng/m3 (range: 0.06-1.24 ng/m3). The mean concentration of benzene in the breathing zone was 41 (SD = 20) micrograms/m3, although a level of 100 micrograms/m3 was slightly exceeded in one subject. In urine samples collected before and after workshifts, two biological indicators of exposure to benzene were measured, urinary benzene and urinary trans, trans-muconic acid (MA). The mean values of urinary benzene before and after workshift were similar (98, SD = 81 and 83, SD = 55 ng/l; n = 63; Wilcoxon's T-test = not significant), while a moderate increase in the metabolite was observed (MA = 0.08, SD = 0.11; 0.11, SD = 0.09 mg/g creatinine, in pre- and post-shift samples respectively; Wilcoxon's T-test, z = 3.00; p < 0.01). The levels of exposure to dusts and 1-NP deriving from diesel engine emissions were comparable to those of other occupational groups with this type of risk (garage mechanics, workers operating diesel engine machinery, etc.). Traffic police exposure to benzene was similar to that of the whole population of Padova (40 micrograms/m3, mean annual 24-hour value). However, the values of urinary MA, like those reported by other authors for non-smoker controls, increased after the workshift, indicating low occupational exposure to this pollutant. It should be noted that traffic police exposure to benzene is much lower than that of other occupational categories, e.g., fuel pump distributors.

[Organization of health and safety activities in health care centers]. / Organizzazione delle attività di prevenzione e sicurezza nelle aziende sanitarie.

The paper proposes a model for health and safety organization in health care units and hospitals which takes account of the risk assessment procedures required by law and the quality assessment of the measures thus taken. A redefinition is given of the role of Medical Director and of the functions, aims and standards on which health and safety service and the services of an authorized occupational health physicians must be based.

A pleiotropic Arabidopsis thaliana mutant with inverted root chirality.

Circumnutation is an oscillating movement of a growing plant organ that is believed to result from an endogenous rhythmic process intrinsic to growth. Circumnutating organs, as they extend, describe a helical trace. In Arabidopsis thaliana (L.) Heynh. circumnutation is particularly evident in primary roots and occurs, as in most plants, in a right-handed direction when viewed from above in the direction of the growing tips. We have discovered a pleiotropic mutant of Arabidopsis with left-handed root circumnutation. Major abnormalities of the mutant are: (i) a reduced size of all organs, mainly due to a defect in cell elongation or expansion; (ii) a zigzagging pattern of stem pith cells, reminiscent of the "erectoides" phenotype of the lk mutant of Pisum; (iii) roots of the mutant are gravitropic but as they grow, they form tight, left-handed coils. Genetically, the mutant depends on the presence of two independent monogenic recessive factors acting additively. The mutant alleles of both factors alter the growth of the aerial organs in a similar manner but differ at the root level: one mainly produces non-circumnutating roots, the other changes the direction of circumnutation from right to left hand.

Linkage relationships between regulatory and structural gene loci involved in zein synthesis in maize.

The synthesis of at least 15 zein polypeptides is under the control of regulatory gene loci. One of these, Opaque-2 (chromosome 7, position 16) strongly reduces the zein accumulation without modifying the zein molecular components. The linkage relationship between the regulatory gene 02 and the 5 structural loci (Zp1, Zp2, Zp3, Zp6, Zp12) segregating with sample Mendelian ratios have been studied. Zp1, Zp2, Zp3 are closely linked to each other; moreover this gene cluster is located on chromosome 7 at 5.5 cM from the Opaque-2 locus. The structural loci Zp6 and Zp12 are not linked with each other, with the 02 locus or with Zp1, Zp2, Zp3. From our data it follows that the zein structural genes are located in at least three positions on the maize genome. The scattering in the genome of the genes controlled by the Opaque-2 locus suggests a transacting role for this regulatory element.

Heterogeneity of storage proteins in maize.

The extensive charge heterogeneity of maize (Zea mays L.) zeins observed in isoelectric focusing (IEF) (about 15 bands with pI's in the pH range 6-9) has been found to be independent of extraction procedures or of endosperm development. Zeins do not stain for glycoproteins and exhibit only one lipoprotein component, with pI 3, representing 3-5% of the total protein.Zeins are very resistant to in vitro deamidation, at both acidic and alkaline pH, at high temperatures, and for rather prolonged times. On the basis of the zein content in acidic and basic amino acids, and of the respective pI's exhibited in IEF (mostly in the pH range 7-8) it has been calculated that at least 90% of the glutamic and aspartic acids (≈52 residues out of a total of ≈ 190) are present as asparagine and glutamine.Amino acid analysis of zein fractions isolated by preparative IEF has demonstrated changes in the composition of 18 amino acid residues. However, since these changes affect only neutral and hydrophobic residues, it is concluded that the observed zein heterogeneity is partly based on in vivo deamidation of glutamine and asparagine and partly to spot mutations in some of the genes responsible for zein synthesis.

Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes.

Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.

Genes for zein subunits on maize chromosome 4.

This paper maps nine genes coding for zein subunits on maize chromosome 4. Six of them (Zp6h, Zp10, Zp14, Zp15, Zp22) encode for subunits with a molecular weight of 22 Kd (kilodaltons), while three (Zp27, Zp28, Zp30) code 20-kd subunits. The six 22-kd related genes are not contiguous but are scattered on both chromosome arms, whereas Zp27, Zp28, and Zp30 are more tightly linked in the chromosome short arm in a segment 5 crossover units long. The organization of zein genes on chromosome 4 shows a close analogy with that of zein loci on chromosome 7. This suggests that both maize chromosomes evolved by duplication of short segments.

Zein level in maize endosperm depends on a protein under control of the opaque-2 and opaque-6 loci.

Zeins, the major endosperm proteins of maize, represent about 50% of total seed proteins and consist of several alcohol-soluble polypeptides encoded by at least four families of genes. The accumulation of zeins is under the control of several broadly defined regulatory genes. One of these, the opaque-2 mutant, lowers the level of zeins, thus improving the nutritional quality of maize meals. We demonstrate here that a salt-soluble protein of molecular weight 32,000 (b-32) is under control of O2. Seven o2 recessive alleles are CRM- when assayed with b-32 antibody. The O2 gene does not encode protein b-32, which apparently is the gene product of the O6 locus. 06 endosperms are CRM- for b-32 protein and are almost devoid of zeins. This indicates that b-32 plays an important role in accumulation of zeins.