Polyclonal antibodies to a recombinant coat protein of Potato virus A.

Specific mouse antibodies against a recombinant coat protein (CP) of Potato virus A (PVA) were produced. The PVA CP gene was cloned in an expression vector pMPM4omega. After expression in Escherichia coli the presence of the expressed CP was proved by Western blot analysis using polyclonal and monoclonal antibodies (MAbs). The expressed CP was purified by centrifugation in CsCl density gradient or on a sucrose cushion. The production of virus-like particles (VLPs) was proved by electron microscopy. The purified CP was used for preparation of a mouse antiserum which had a titer of 1:1024 in ELISA and reacted specifically in Western blot analysis and indirect plate-trapped antigen ELISA (PTA-ELISA).

Monitoring the genotoxicity of soil extracts from two heavily polluted sites in Prague using the Tradescantia stamen hair and micronucleus (MNC) assays.

We have taken soil samples from two sites in Prague, the capital of the Czech Republic, that are heavily polluted by motor vehicles. As a negative control, soil samples from a recreational site in Prague with no motor vehicle traffic were used. Soil samples from these sites were extracted with water or 5% dimethylsulphoxide (DMSO) for 24 h and cuttings of Tradescantia clone 4430 were immersed for 12 h at 25 degrees C in the extraction solutions. As a positive control Tradescantia plants have been treated with the promutagenic arylamine o-phenylenediamine at the same treatment conditions. None of the tested soil extractions significantly increased the frequency of somatic mutations in the stamen hair assay. By contrast, a 5% DMSO soil extract from one of the tested sites (entrance of the Letná tunnel) significantly increased the frequency of micronuclei (MNC) in the pollen mother tetrad cells. A repetition of the treatment 14 days later also resulted in an increase in the frequency of MNC, however the increase was not statistically significant. This study was conducted for the International Programme on Plant Bioassays.

[Evaluation of diet in a sample of Czech mothers six months after delivery]. / Zhodnocení stravování vzorku ceských matek v sestém mesíci po porodu.

BACKGROUND: Health care of nursing mothers and their infants is an important priority of primary preventive care. The mother's diet plays an important role in this respect. The objective of the presented investigation was to assess the adequacy of the dietary intake of lactating mothers during the sixth month after delivery. METHODS AND RESULTS: Data on the education, body weight, height of the mother, dietary intake, evaluated from a three-day dietary record, were collected from 131 nursing mothers and compared with the Czech recommended dietary allowances for nursing mothers as well as with data from 265 controls, i.e. women who did no longer breastfeed their babies. From the results ensues a significantly higher calcium intake (937 mg; SD = 415, p < 0.001), vitamin B1 (1.1 mg, SD = 0.5, p < 0.001), total energy (8.7 MJ, SD = 2.6, p < 0.01), protein 75.4 g, SD = 18, p < 0.01), carbohydrates (281 g, SD = 112, p < 0.01) and riboflavin (1.3 mg, SD = 0.5, p < 0.01) in nursing women as compared with those not nursing. The nursing mothers, however, do not meet the Czech recommended dietary allowances as regards total energy, calcium, vitamin C, linoleic acid, vegetable proteins and iron. Other problematic nutrients-magnesium, zinc, folates, pyridoxine, selenium and iodine could not be assessed as they are not listed in the Czech food composition tables. In women with university education the energy and nutrient intake was in the majority more favourable than in women with elementary education. On the other hand no statistical differences were found in weight increments during the period from the beginning of gestation to the sixth month after delivery between nursing and not nursing mothers. CONCLUSIONS: The results of analysis of the dietary intake of nursing mothers indicate that the Czech recommended allowances are not met as regards energy, calcium, linoleic acid, protein and iron. Whether the intake is really inadequate or whether the recommended allowances are excessive remains an open question.

Mutagenic synergy between paraoxon and plant-activated m-phenylenediamine or 2-acetoxyacetylaminofluorene.

Paraoxon (diethyl-p-nitrophenylphosphate) is the toxic, but non-mutagenic metabolite of the organophosphorus ester insecticide parathion. Although this agent has been used as a deacetylase inhibitor in many studies, we discovered a mutagenic synergy when paraoxon was incubated with plant-activated m-phenylenediamine (mPDA) or with direct-acting 2-acetoxyacetylaminofluorene (2AAAF) and S. typhimurium tester strains. Using non-toxic concentrations of plant-activated mPDA and paraoxon a 10-fold increase in the mutant yield of S. typhimurium was observed. The mutagenicity of the plant-activated mPDA product required that O-acetyltransferase (OAT) be expressed by the S. typhimurium tester strain. However, the paraoxon-dependent mutagenic synergy was observed using the direct-acting arylamine metabolite, 2AAAF, with strains YG1024, TA98 and TA98/1,8-DNP6 regardless of their OAT activity. This mutagenic synergy is dependent upon the presence of an activated acetylated form of the arylamine. The data presented here demonstrate that this mutagenic synergy is limited to paraoxon and not to the parent compound (parathion) or to a major metabolite of parathion (p-nitrophenol).

Environmental monitoring for genotoxicity with plant systems. An introduction and study design.

Under the sponsorship of the International Programme on Chemical Safety (IPCS), 17 laboratories from diverse regions of the world participated in evaluating the utility of four plant bioassays for detecting genetic hazards of environmental chemicals. The bioassays included in this collaborative study were: Arabidopsis thaliana embryo and chlorophyll assay and Tradescantia stamen hair assay, Tradescantia paludosa micronucleus assay and Vicia faba root tip assay. Four to six laboratories participated in the performance of each of the bioassays. All laboratories participating in a particular bioassay were supplied with uniform plant material as well as standardized protocol. Five direct acting water soluble test chemicals, i.e. maleic hydrazide, methyl nitrosourea, ethyl methanesulfonate, sodium azide and azidoglycerol, were selected for this study. The study was designed to be completed in three phases. Ethyl methanesulfonate was used as a positive control and has already been reported earlier (Sandhu et al., 1991). The data from the remaining four chemicals used for the evaluation of four plant test systems in the first phase of the collaborative study are reported in this issue.

Arabidopsis assay for mutagenicity.

Four laboratories, two in the Czech Republic (Brno and Prague) and two in the CIS (Moscow and Duschanbe), participated in the International Programme on Chemical Safety's (IPCS) collaborative study to evaluate the utility of the most commonly used plant test systems, including the Arabidopsis thaliana assay, for assessing the mutagenic potential of environmental agents. Out of the five compounds evaluated in the Arabidopsis assay, three compounds, i.e., ethyl methanesulfonate, N-methyl-N-nitrosourea, and azidoglycerol, were reported to be mutagenic by all four participating laboratories. Sodium azide (NaN3) demonstrated a negative response in all four laboratories, whereas maleic hydrazide was reported to be weakly mutagenic by one laboratory and nonmutagenic by the other three laboratories.

Environmental monitoring for genotoxicity with plant systems. Results and recommendations.

In the first phase of a collaborative study by the International Programme on Chemical Safety (IPCS), four coded chemicals, i.e. azidoglycerol (AG, 3-azido-1,2-propanediol), methyl nitrosourea (MNU), sodium azide (NaN3) and maleic hydrazide (MH), and ethyl methanesulfonate (EMS) as a positive control were tested in four plant bioassays, namely the Arabidopsis embryo and chlorophyll mutation assay, the Tradescantia stamen hair assay (Trad-SH assay), the Tradescantia micronucleus assay (Trad-MCN), and the Vicia faba root tip assay. Seventeen laboratories from diverse regions of the world participated with four to six laboratories each using one plant assay. For the Arabidopsis assay, laboratories were in agreement with MNU and AG giving positive responses and NaN3 giving a negative response. With the exception of one laboratory which reported MH as weakly mutagenic, no mutagenic response was reported for MH by the other laboratories. For the Vicia faba assay, all laboratories reported a positive response for MNU, AG, and MH, whereas two of the six laboratories reported a negative response for NaN3. For the Trad-SH assay, MH was reported as giving a positive response and a positive response was also observed for MNU with the exception of one laboratory. NaN3, which exhibited a relatively high degree of toxicity, elicited a positive response in three of the five laboratories. AG was found positive in only one of the two laboratories which tested this chemical. For the Trad-MCN assay, MNU and MH were reported as positive by all laboratories, while four out of five laboratories reported NaN3 to be positive. Only one of three laboratories reported AG to be positive. The major sources of variability were identified and considered to be in the same range as found in similar studies on other test systems. Recommendations were made for minor changes in methodology and for initiating the second phase of this study.

An E. coli ada transgenic clone of Nicotiana tabacum var. Xanthi has increased sensitivity to the mutagenic action of alkylating agents, maleic hydrazide and gamma-rays.

Two transgenic clones X3 and X15 of Nicotiana tabacum var. Xanthi, heterozygous in two genes (a1 and a2) for chloroplast differentiation and transformed with the E. coli DNA repair gene ada cloned downstream from the 1' direction of the dual mas promoter, differed in the expression of the ada gene, in the number of copies of integrated T-DNA and in the response to the mutagenic action of alkylating and non-alkylating agents. The X3 genome contained four copies and the X15 genome one copy of T-DNA, nevertheless the expression of the ada gene, measured by the activity of O6-alkylguanine DNA alkyltransferase (ATase), was about six times higher in X15 than in X3. ATase activity in both clones was highest in extracts from callus whereas very low (X15) or no (X3) activity was detected in leaf extracts. This may explain the lack of difference between X15 and non-transformed tobacco (NTX) in the frequency of N-methyl-N-nitrosourea (MNU)-induced somatic mutations in leaves. In contrast, the frequency of somatic mutations in X3 was about 2-5 times higher than in NTX and X15 after the same doses of MNU, methyl methanesulfonate, maleic hydrazide and gamma-rays. Alteration of plant gene(s) essential in mutation pathway(s) by insertion of T-DNA or by somaclonal variation may explain the higher sensitivity of the X3 clone.

Mutagenicity of 3-azido-1,2-propanediol and 9-(3-azido-2-hydroxypropyl)-adenine in repair deficient strains of Escherichia coli.

The mutagenicity of two non-aromatic organic azido compounds, 3-azido-1,2-propanediol (AG) and 9-(3-azido-2-hydroxypropyl)-adenine (AHPA), was studied in E. coli repair deficient strains uvrA6, uvrA6 + umuC36, uvrA6+ umuC122::Tn5, polA1, tagA1+ alkA1, ada and dam3. The mutagenicity of both agents was markedly enhanced by defects of UvrABC excinuclease (uvrA6) and was independent of umuC function of the SOS error-prone pathway. Neither azido compound promoted umuDC operon expression. The mutagenicity of AG in tag A1, alkA1 and ada mutants does not differ from that found in the wild-type strain. The expression of both ada and alkA genes was not elevated by AG. Experiments on polA1 and dam3 mutants suggest that DNA polymerase I as well as the mutHLS mismatch repair pathway does not contribute to the removal of putative DNA lesions induced by AG.

Inhibitory effects of acetaminophen, 7,8-benzoflavone and methimazole towards N-nitrosodimethylamine mutagenesis in Arabidopsis thaliana.

The metabolic inhibitors acetaminophen, 7,8-benzoflavone, and methimazole significantly reduced the mutagenicity of the promutagen N-nitrosodimethylamine in the higher plant Arabidopsis thaliana. In contrast, these metabolic inhibitors had no effect on the mutagenicity of the direct-acting mutagen N-methyl-N'-nitro-N-nitrosoguanidine.

Increased resistance to the toxic effects of alkylating agents in tobacco expressing the E. coli DNA repair gene ada.

The protein coding region of the E. coli gene ada has been transferred to tobacco plants by a leaf disc transformation procedure involving an Agrobacterium tumefaciens Ti plasmid. Transformed plants were shown to be transgenic for the ada message and had increased levels of O6-alkylguanine DNA alkyltransferase activity. The N-methyl-N-nitrosourea- or taurinechlorethylnitrosourea-induced inhibition of growth of calluses or of cells in suspension was considerably lower in ada-transformed than in non-transformed plants. This indicates that O6-alkylguanine, O4-alkylthymine or phosphotriesters are growth-inhibitory lesions in tobacco.

Mutagenic activity of 6-azido deoxyhexoses and azido alcohols in Salmonella typhimurium and its inhibition by a structure-similar carbon source in the medium.

6-Azido-6-deoxy (AZd) derivatives of D-glucose, D-mannose, D-altrose, D-allose, L-idose, D-galactose, D-galactonic acid and D-galactitol, 3-azido-1,2-propanediol (azidoglycerol), 3,1-diazido-2-propanol (diazidoglycerol) and (at much higher doses) 2-azidoethanol were mutagenic in Salmonella typhimurium strains TA100 and TA1535. The mutagenic response was similar to that induced by sodium azide, i.e., the azido compounds failed to induce mutations in strain TA98, and mutagenesis was independent of plasmid pKM101, and independent of external activation. The specific mutagenicity (his+ rev/mmole) of AZd-glucose and AZd-galactose was decreased with increasing concentrations of D-glucose or D-galactose in the minimal agar medium and enhanced 100-fold or more when 0.2% citrate rather than 0.2% glucose served as the carbon source in the medium. Similarly, the mutagenic efficiency of azidoglycerol was inhibited by glycerol but not by D-glucose or D-galactose; however, the mutagenicity of sodium azide was not influenced by any of these carbon sources in the medium. The inhibition of the mutagenic action of azido hexoses and azido alcohols by non-azido structural analogs is assumed to reside in competition in transmembrane transport or for the metabolic pathways.

UV-irradiation potentiates the antimutagenicity of p-aminobenzoic and p-aminosalicylic acids in Salmonella typhimurium.

UV-irradiation (254 nm, 10 or 20 J/cm2) of p-aminobenzoic acid (PABA) and p-aminosalicylic acid (NaPAS) potentiated their antimutagenicity towards N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in Salmonella typhimurium. Their inhibitory action towards the formation of the mutagen N-methyl-N-nitrosourea from the nitrosation mixture of N-methylurea and nitrite was also increased by UV-irradiation. In contrast, UV-irradiated PABA exhibited no inhibitory effects towards the mutagenicity of sodium azide or 3-azidoglycerol. Neither PABA nor NaPAS nor their UV-irradiation products were themselves mutagenic in the Ames assay.

The mutagenicity of azido derivatives of monosaccharides and alcohols and of N-(3-azido-2-hydroxypropyl) derivatives of purines and pyrimidines in Arabidopsis thaliana and in Salmonella typhimurium.

The azido derivatives of alcohols (3-azido-1,2-propandiol and 1,3-diazido-2-propanol) and monosaccharides (6-azido-6-deoxy-beta-D-glucose and 6-azido-6-deoxy-beta-D-galactose), as well as the proximal mutagenic product of sodium azide metabolism beta-azido-L-alanine, exhibited a high mutagenic activity in a higher plant Arabidopsis thaliana and in Salmonella typhimurium. In contrast, 11 N-(3-azido-2-hydroxypropyl) derivatives of purines and pyrimidines (adenine, thymine, uracil, cytosine, 2-amino-6-chloropurine, 6-chloropurine, 2,6-diaminopurine, 6-methylthiopurine, 4-O-methylthymine, 4-O-methyluracil and 7-deaza-8-azaadenine) were mutagenic in the Ames assay but ineffective in the Arabidopsis mutagenicity assay.

[Microcirculation in the subcutaneous layer and in muscles in diabetes mellitus]. / Mikrocirkulácia v podkozí a vo svale pri diabetes mellitus.

The work is focused on the possibility of early diagnosis of diabetic microangiopathy. The authors used examination of microcirculation by the method of tissue clearance of Na 131I. In a group of 106 patients with diabetes thus the microcirculation of the lower extremities in the subcutaneous layer and the calf muscle was examined. A group of 36 healthy subjects served as control. An altered microcirculation was found in 75% type 1 diabetics and in all type 2 diabetics. In those with an altered microcirculation only in 23% type 1 diabetics and in 17% type 2 diabetics organ manifestations of diabetic microangiopathy (i.e. retinopathy and nephropathy) were lacking. It seems thus that altered Na 131I absorption in the lower extremities is an earlier sign of microangiopathic affection than identifiable organ changes. Examination of tissue clearance of Na 131I thus could reveal in time individuals threatened by this complication of diabetes.

[Diagnosis of diabetic macroangiopathy of the lower extremities using rheography]. / Diagnostika diabetickej makroangiopatie dolných koncatín pomocou reografie.

Rheography (impedance plethysmography) makes it possible to differentiate the normal condition of arteries of the lower extremity from functional and organic affections. The examination of a group of 325 patients with diabetes mellitus revealed that without the use of rheography, i.e. when relying only on examination of the lower extremities by palpation, the presence of macroangiopathy would escape attention in 46% of the affected subjects. Macroangiopathy of an organic character was found significantly more frequently in type 2 (NIDDM) (35%) than in type 1 (IDDM) (9%; p less than 0.03). Its incidence increased in both types of diabetes with the duration of the disease. Moreover it was significantly associated with hyperlipoproteinaemia. The assembled results drew attention to the great diagnostic value of rheography. It permits also to document objectively the effect of treatment focused on macroangiopathy.

Effects of humic acids, para-aminobenzoic acid and ascorbic acid on the N-nitrosation of the carbamate insecticide propoxur and on the mutagenicity of nitrosopropoxur.

Nitrosation of the carbamate insecticide propoxur at pH 3 and 37 degrees C was determined colorimetrically and found to be time- and sodium nitrite concentration-dependent. Nitrosated propoxur was mutagenic when exposed to the seeds of the higher plant Arabidopsis thaliana but the formation of nitrosopropoxur, the presumed mutagen, was inhibited by humic acids, para-aminobenzoic acid and ascorbic acid. These agents also reduced the mutagenicity of preformed nitrosopropoxur.

Repair of bleomycin-induced DNA double-strand breaks in Vicia faba.

As detected by neutral DNA elution, bleomycin induced at the concentrations tested (5, 10 and 50 micrograms/ml) DNA double-strand breaks (dsbs) in in vitro cultured embryos of V. faba. Most of these breaks were repaired during a 4-h incubation period after treatment. Dsbs also occurred after treatment with 2.5 and 5 mM of N-methyl-N-nitrosourea (MNU) but in contrast to those induced by bleomycin, these dsbs remained unrepaired during the 4-h incubation period following the treatment.