Developmental lead exposure in rats: is a behavioral sequel extended at F2 generation?

Lead toxicity was studied in rats exposed from conception until weaning and assessed by monitoring offspring behavior in both the open field and elevated plus maze and by determining tissue lead in an assessment schedule extended to first (F1) and second (F2) generations. Dams utilized for the F1 generation were submitted to 750 ppm of lead (acetate) in drinking water during pregnancy and lactation. For F1 pups, behavioral alterations were not detected in the elevated plus maze, while in the open field, spontaneous locomotor activity as well as time of both grooming and rearing increased, while freezing time decreased in 30- and 90-day-old rats. Lead content was higher in tissues of 1- and 30-day-old pups. However, in 90-day-old rats, lead was detected only in the femur. F2 generation was lead-free but still presented alterations in both locomotor activity and grooming in 30- and 90-day-old pups. It appears that developmental lead exposure may cause behavioral effects during the developmental stage of the F1 generation, which remains throughout the animal's adult life as a sequel, regardless of lead accumulation, and is extended to the F2 generation of rats. (C) 2001 Elsevier B.V. All rights reserved.

Estudo da atuação dos metais zinco e cádmio sobre os aspectos reprodutivos da espécie Ceriodaphnia dubia geração parental e F1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Generation of a NG2-EYFP mouse for studying the properties of NG2-expressing cells

A range of vectors were made in which the EYFP gene or the Cre gene were inserted in the start codon of the NG2 gene. The NG2-EYFP vectors were used to generate NG2-EYFP “knockin” mice by homologous recombination. The F1 generation showed lack of EYFP expression, due to NeoR cassette interference. Excision of the NeoR, by breeding the F1 generation to ELLA-Cre mice allowed proper expression of EYFP. NG2-EYFP heterozygous mice were characterized in detail for astrocytic, neurogenic and oligodendrocytic properties through antibody labeling. NG2-EYFP+ cells did not label for the astrocyte marker GFAP, but some cells did express S100 Beta. The cells did not label with any neuronal markers like Beta III tubulin, Neun, and double cortin, but many of the NG2-EYFP+ cells made intimate contacts to the neurons. These contacts are widespread throughout the grey and white matter of the brain. The NG2-EYFP+ cells did label for oligodendrocyte markers like PDGFα-R, NG2, Olig2, O4, and Sox 10. There were a few cells termed phantom cells that did label for NG2, but had no EYFP expression. This could have been caused by improper excision of the NeoR cassette in the F2 generation. The heterozygous mouse is a tool to allow the characterization of the in vivo properties of the NG2+ cells. Breeding of these mice to homozygosity yielded an NG2-knockout mouse, which was also subjected to initial characterization. The NG2-EYFP homozygous showed equivalent cell labeling results to the NG2-EYFP heterozygous mouse, but the phantom cells disappeared in the knockout. The results show that the NG2 cells are a heterogenous population that does not express astrocytic or neuronal markers. The homozygous mouse is an ideal tool to further dissect the properties of the cells, lacking NG2 in vivo.

Progeny evaluation for resistance to Phaeosphaeria leaf spot in tropical maize

Maize breeding programmes in Brazil and elsewhere seek reliable methods to identify genotypes resistant to Phaeosphaeria leaf spot. The area under the disease progress curve (AUDPC) is an accurate method to evaluate the severity of foliar diseases. However, at least three data points are required to calculate the AUDPC, which is unfeasible when there are thousands of genotypes to be assessed. The aim of this work was to estimate the heritability of disease resistance, evaluate disease severity at different times using a nine-point scale in comparison to the AUDPC, and establish the most suitable phenological period for disease assessment. A repeated experiment was conducted in a 11 x 11 lattice experimental design with three replications. Disease assessments were carried out at flowering, 15 and 30 days post-anthesis for the parental lines DS95, DAS21, the F1 generation and 118 F2:3 progenies. Then, the AUDPC was obtained and results compared with the single-point evaluations used to calculate it. Individual and joint analyses of variance were conducted to obtain heritabiliy estimates. The assessments performed after the flowering stage gave higher estimates of heritability and correlation with AUDPC. We concluded that one assessment between the 15th and 30th day after flowering could provide enough information to distinguish maize genotypes for their resistance to Phaeosphaeria leaf spot under tropical conditions.

Mice expressing the E7 oncogene of HPV16 in epithelium show central tolerance, and evidence of peripheral anergising tolerance, to E7-encoded cytotoxic T-lymphocyte epitopes

In order to derive mice which expressed both the E7 open reading frame transgene of human papillomavirus type 16 in skin and MHC class 1 restriction elements for several E7-encoded cytotoxic T-lymphocyte (CTL) epitopes, K14.HPV16E7 mice which express E7 in basal keratinocytes were crossed to the F1 generation with A2.1 K-b transgenic mice which express the MHC binding cleft domains of human HLA A*0201, and murine H-2(b). F1 mice (denoted K14E7xA2.1) expressed E7 in the thymus at least as early as 2-5 days before birth. Immunisation of FVBxA2.1 control mice (transgenic for HLA A*0201 and H-2(b) but not for E7), with two HLA A*0201-restricted epitopes of E7 and one H-2(b)-restricted CTL epitope of E7, gave strong primary CTL responses recognising epitope-pulsed or constitutively E7-expressing syngeneic target cells. In contrast, in immunised K14E7xA2.1 mice, the CTL responses to the H-2(b) epitope and one of the HLA A*0201 CTL epitopes were strongly down-regulated, and to the other HLA A*0201 epitope, completely abolished, as demonstrated by percentage specific killing by bulk splenocyte cultures in cyrotoxicity assays, and by CTL precursor frequency analysis, In thymus-transplanted bone marrow radiation chimeras in which the immune system of K14E7xA2.1 mice was replaced by a FVBxA2.1 immune system, specific immunisation did not result in reemergence of strong E7-directed CTL responses. In agreement with these in vitro findings, specific immunisation failed to significantly alter the course of E7-associated tumour development in K14E7xA2.1 mice. These data are consistent with a model of central deletional CTL tolerance to E7-encoded epitopes recognised in the context of two distinct MHC class 1 restriction elements, and with the possibility of peripheral T-cell anergy maintained by expression of E7 in the skin. (C) 1998 Academic Press.

Potential of biologically active plant oils to control mosquito larvae (Culex pipiens, Diptera: Culicidae) from an egyptian locality

The insecticidal effect of six commercially available plant oils was tested against 4th larval instars of Culex pipiens. Larvae were originally collected from Meit El-Attar, Qalyubia Governorate, Egypt, and then reared in the laboratory until F1 generation. The LC50 values were 32.42, 47.17, 71.37, 83.36, 86.06, and 152.94 ppm for fenugreek (Trigonella foenum-grecum), earth almond (Cyperus esculentus), mustard (Brassica compestris), olibanum (Boswellia serrata), rocket (Eruca sativa), and parsley (Carum ptroselinum), respectively. The tested oils altered some biological aspects of C. pipiens, for instance, developmental periods, pupation rates, and adult emergences. The lowest concentrations of olibanum and fenugreek oils caused remarkable prolongation of larval and pupal durations. Data also showed that the increase of concentrations was directly proportional to reduction in pupation rates and adult emergences. Remarkable decrease in pupation rate was achieved by mustard oil at 1000 ppm. Adult emergence was suppressed by earth almond and fenugreek oils at 25 ppm. In addition, the tested plant oils exhibited various morphological abnormalities on larvae, pupae, and adult stages. Consequently, fenugreek was the most potent oil and the major cause of malformation of both larval and pupal stages. Potency of the applied plant oils provided an excellent potential for controlling C. pipiens.

Repeated unidirectional introgression towards Populus balsamifera in contact zones of exotic and native poplars.

As the evolutionary significance of hybridization is largely dictated by its extent beyond the first generation, we broadly surveyed patterns of introgression across a sympatric zone of two native poplars (Populus balsamifera, Populus deltoides) in Quebec, Canada within which European exotic Populus nigra and its hybrids have been extensively planted since the 1800s. Single nucleotide polymorphisms (SNPs) that appeared fixed within each species were characterized by DNA-sequencing pools of pure individuals. Thirty-five of these diagnostic SNPs were employed in a high-throughput assay that genotyped 635 trees of different age classes, sampled from 15 sites with various degrees of anthropogenic disturbance. The degree of admixture within sampled trees was then assessed through Bayesian clustering of genotypes. Hybrids were present in seven of the populations, with 2.4% of all sampled trees showing spontaneous admixture. Sites with hybrids were significantly more disturbed than pure stands, while hybrids comprised both immature juveniles and trees of reproductive age. All three possible F1s were detected. Advanced-generation hybrids were consistently biased towards P. balsamifera regardless of whether hybridization had occurred with P. deltoides or P. nigra. Gene exchange between P. deltoides and P. nigra was not detected beyond the F1 generation; however, detection of a trihybrid demonstrates that even this apparent reproductive isolation does not necessarily result in an evolutionary dead end. Collectively, results demonstrate the natural fertility of hybrid poplars and suggest that introduced genes could potentially affect the genetic integrity of native trees, similar to that arising from introgression between natives.

Biology of Triatoma klugi Carcavallo, Jurberg, Lent & Galvão 2001 (Heteroptera: Reduviidae) under Laboratory Conditions: Effects of Distinct Blood Sources and Susceptibility to Trypanosoma cruzi and Trypanosoma rangeli

The life cycle of Triatoma klugi Carcavallo, Jurberg, Lent & Galvão 2001 was compared under laboratory conditions using two groups of the F1 generation obtained from field-collected bugs. Among the 100 nymphs weekly fed on mice (Group A) or chicken (Group B), 77% of Group A and 67% of Group B reached the adult stage, and the mean time from the first nymphal stage to adult was 190.08 ± 28.31 days and 221.23 ± 40.50, respectively. The average span in days for each stage per group and the number of blood meals required for each stage were also evaluated. The overall mortality rate was 23% and 33% for Groups A and B, respectively. The mean number of eggs laid per month in a three-month period was of 56.20, 51.70 and 73.20 for Group A, and 64.50, 53.50 and 38.71 for Group B. Despite the blood source, comparative analysis revealed no statistically significant differences in the life cycle of T. klugi under laboratory conditions. Infection rates over 60% were observed for both Trypanosoma cruzi strains tested. Even revealing high infection rates of the hemolymph by T. rangeli strains, T. klugi revealed no salivary gland infections and was not able to transmit the parasite.

Susceptibility of Anopheles aquasalis and An. darlingi to Plasmodium vivax VK210 and VK247

The susceptibility of Anopheles aquasalis (F3 generation) and An. darlingi (F1 generation) to Plasmodium vivax circumsporozoite protein phenotypes from a limited number of blood samples of malaria patients in Belém, state of Pará, Brazil, was examined. A polymerase chain reaction was used to determine the P. vivax phenotypes in blood samples and the blood-fed infected mosquitoes were dissected and tested by ELISA. In all patient infections, more infected An. aquasalis and An. darlingi were positive for VK210 compared with VK247.

Biomphalaria tenagophila: dynamics of populations of resistant and susceptible strains to Schistosoma mansoni, with or without pressure of the parasite

Resistant (Taim, RS) and susceptible albino (Joinville, SC) Biomphalaria tenagophila populations were kept together, at different proportions, throughout a 18-month-period. Some of the snail groups were submitted to Schistosoma mansoni infection. The targets of this study were (a) to analyze the populational dynamics among resistant and susceptible individuals to S. mansoni; (b) to study the resistance phenotype in descendants of cross-breeding; (c) to observe whether the parasite could exert any kind of selection in those snail populations. Throughout the experiment it could be observed that the susceptible B. tenagophila strain (Joinville) underwent a selective pressure of the parasite that was negative, since the individuals showed a high mortality rate. Although B. tenagophila (Taim) population presented a higher mortality rate without pressure of the parasite, this event was compensated by a reproductive capacity. B. tenagophila Taim was more fecund than B. tenagophila Joinville and was able to transmit the resistance character to their descendants. F1 generation obtained by cross-breeding between resistant and susceptible lineages was completely resistant to S. mansoni infection, irrespective of the Taim proportion. Moreover, less than 5% of F2 progeny were susceptible to S. mansoni infection.

PCR-RAPD and PCR-RFLP polymorphisms detected in Anopheles cruzii (Diptera, Culicidae)

The aim of the present study was to examine genetic variability in populations of An. cruzii by employing PCR-RAPD and PCR-RFLP markers. All analyses were carried out using individuals of the F1 generation of wild caught females obtained in Santa Catarina State (Florianópolis and São Francisco do Sul), Paraná State (Morretes, Paranaguá and Guaratuba) and São Paulo State (Cananéia). In the PCR-RAPD experiments, seven primers were used for comparisons within and among populations. The restriction profile of the ITS2 including a fragment of both 5.8S and 28S regions of the rDNA was obtained with the enzymes BstUI, HaeIII, TaqI, HhaI, Sau96I, HinfI, HincII and NruI. The PCR-RAPD method detected a large number of polymorphic bands. Genetic distance among populations of An. cruzii varied from 0,0214 to 0,0673, suggesting that all individuals used in the analyses belong to a single species. The number of migrants per generation (Nm) was 4.3, showing the existence of gene flow among populations. The restriction profile of the ITS2, 5.8S and 28S gene regions was similar in all An. cruzii samples, whereas the results obtained by using HhaI and NruI are indicative that the individuals analyzed have nucleotide sequences distinct from those of An. cruzii samples from Peruíbe and Juquiazinho deposited in GenBank.

Crocidura cossyrensis Contoli, 1989 (Mammalia, Soridiae): karyotype, biochemical genetics and hybridization experiments

Crocidura cossyrensis Contoli, 1989 (Mammalia, Soricidae): karyotype, biochemical genetics and hybridization experiments. - The shrew Crocidura cossyrensis Contoli, 1989 from Pantelleria (I), a Mediterranean island 100 km south of Sicily and 70 km west from Tunisia, was investigated in order to understand its origin and its relationship with C. russula from Tunisia, Morocco and Switzerland. With the exception of a single heterozygote centric fusion, C. cossyrensis had a karyotype identical with that of C russula from Tunisia (2N = 42, NF = 70 to 72), but it differed from C russula from Morocco and Switzerland (2N = 42, NF = 60). The former have 5-6 pairs of chromosomes with small arms that are acrocentric in the latter. Genetic comparisons with allozyme data revealed small genetic distance (0.04) between C cossyrensis and C russula from Tunisia. In contrast, this eastern clade (Tunisia and Pantelleria) is separated from the western clade (Switzerland and Morocco) by a genetic distance of 0.14. A hybridization experiment between shrews from Pantelleria and Switzerland lead rapidly to an F1 generation. From 12 F1 hybrids that were backcrossed, females reproduced normally, but none of the males did so. Concluding from the results, C. cossyrensis from Pantelleria and C. russula cf. agilis from Tunisia belong to the same taxon that may have reached the differentiation of a biological species within the C. russula group. More geographic samples are needed to determine the definitive taxonomic positions of these shrews.

Interference of genotypes x environments interaction in the genetic control of resistance to Asian rust soybean

The objectives of this work were to identify parents resistant to Asian soybean rust using diallel crosses, obtain information on the genetic control of soybean resistance to the pathogen and verify whether the combining ability estimates interact with the environment (year or time of assessment). The F1 generation was obtained in a greenhouse from crosses between five contrasting parents for the trait resistance to soybean rust, in a complete diallel without reciprocals. Two rust-severity assessments were carried out on individual soybean plants of 25 treatments (parents and F2 and F3 populations) in 2006/2007 and 2007/2008, in an experimental field at Embrapa Soja, Londrina, PR, Brazil. Additive effects predominated in the genetic control of soybean resistance to Asian rust, and the interaction of the segregant populations with the environment, although significant, did not alter the genetic parameter's general combining ability (GCA) and specific combining ability estimates, indicating that estimates obtained in one year and one assessment can be extrapolated to others. BR01-18437 inbred line is resistant to Asian rust and showed high GCA effects. This line should be used as parent if the objective is the resistance to Phakopsora pachyrhizi.

Characterization of Leaf-Type Ferredoxin-NADP+ Oxidoreductase (FNR) Isoforms in Arabidopsis thaliana

Life on earth is based on sunlight, which is captured in chemical form by photosynthetic reactions. In the chloroplasts of plants, light reactions of photosynthesis take place at thylakoid membranes, whereas carbon assimilation reactions occur in the soluble stroma. The products of linear electron transfer (LET), highly-energetic ATP molecules, and reducing power in the form of NADPH molecules, are further used in the fixation of inorganic CO2 molecules into organic sugars. Ferredoxin-NADP+ oxidoreductase (FNR) catalyzes the last of the light reactions by transferring electrons from ferredoxin (FD) to NADP+. In addition to LET, FNR has been suggested to play a role in cyclic electron transfer (CET), which produces ATP without the accumulation of reducing equivalents. CET is proposed to occur via two putative routes, the PGR5- route and the NDH-route. In this thesis, the leaf-type FNR (LFNR) isoforms LFNR1 and LFNR2 of a model organism, Arabidopsis thaliana, were characterized. The physiological roles of LFNRs were investigated using single and double mutant plants. The viability of the single mutants indicates functionality of both isoforms, with neither appearing to play a specific role in CET. The more severe phenotype of low-temperature adapted fnr2 plants compared to both wild-type (WT) and fnr1 plants suggests a specific role for LFNR2 under unfavorable growth conditions. The more severe phenotype of the fnr1 x fnr2 (F1 generation) plants compared to single mutants reflects down-regulated photosynthetic capacity, whereas slightly higher excitation pressure indicates mild over-excitation of electron transfer chain (ETC). However, induction of CET and various photoprotective mechanisms enable adaptation of fnr1 x fnr2 plants to scarcity of LFNR. The fnr1 fnr2 plants (F2 generation), without detectable levels of LFNR, were viable only under heterotrophic conditions. Moreover, drought stress induced acceleration of the rate of P700 + re-reduction in darkness was accompanied by a concomitant up-regulation of the PGR5-route specific components, PGR5 and PGRL1, demonstrating the induction of CET via the PGR5-route. The up-regulation of relative transcriptional expression of the FD1 gene indicates that the FD1 isoform may have a specific function in CET, while no such role could be defined for either of the LFNR isoforms. Both the membrane-bound and soluble LFNR1 and LFNR2 each appear as two distinct spots after 2D-PAGE with different isoelectric points (pIs), indicating the existence of post-translational modifications (PTMs) which do not determine the membrane attachment of LFNR. The possibility of phosphorylation and glycosylation PTMs were excluded, but all four LFNR forms were shown to contain acetylated lysine residues as well as alternative N-termini. N-terminal acetylation was shown to shift the pI of both LFNRs to be more acidic. In addition, all four LFNR forms were demonstrated to interact both with FD1 and FD2 in vitro

Effect of obesity on rat reproduction and on the development of their adult offspring

The aim of the present study was to assess the reproductive parameters of obese Wistar rats and to determine the frequency of their obese adult offspring. Neonatal rats were divided into two groups: F1 generation, induced to obesity by monosodium glutamate (MSG; F1MSG, N = 30), and rats given saline (F1CON, N = 13). At 90 days of age all animals were mated, producing the F2 offspring (F2CON, N = 28; F2MSG, N = 15). Reproductive parameters (fertility, pregnancy, and delivery indexes) were evaluated in F1 rats. F2 newborns were weighed, and the obesity parameter for F1 and F2 generations was determined from months 5 to 7 of life. At month 7, periovarian fat was weighed and no differences were found. Mean newborn weight also did not differ. The F1 and F2MSG groups presented approximately 90% of obese rats since month 5 of life, whereas F1 and F2CON groups presented only 33%. There was no difference in periovarian weight among groups. Although obesity did not affect reproductive parameters, obese dams (F1MSG) were responsible for the appearance of obesity in the subsequent generation. Thus, obesity induced by neonatal MSG administration did not interfere with reproduction, but did provide a viable model for obesity in second-generation adult Wistar rats. This model might contribute to a better understanding of the pathophysiological mechanisms involved in transgenerational obesity.