Generation and characterization of a humanized PPARδ mouse model.

BACKGROUND AND PURPOSE: Humanized mice for the nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ), termed PPARδ knock-in (PPARδ KI) mice, were generated for the investigation of functional differences between mouse and human PPARδ and as tools for early drug efficacy assessment. EXPERIMENTAL APPROACH: Human PPARδ function in lipid metabolism was assessed at baseline, after fasting or when challenged with the GW0742 compound in mice fed a chow diet or high-fat diet (HFD). KEY RESULTS: Analysis of PPARδ mRNA levels revealed a hypomorph expression of human PPARδ in liver, macrophages, small intestine and heart, but not in soleus and quadriceps muscles, white adipose tissue and skin. PPARδ KI mice displayed a small decrease of high-density lipoprotein-cholesterol whereas other lipid parameters were unaltered. Plasma metabolic parameters were similar in wild-type and PPARδ KI mice when fed chow or HFD, and following physiological (fasting) and pharmacological (GW0742 compound) activation of PPARδ. Gene expression profiling in liver, soleus muscle and macrophages showed similar gene patterns regulated by mouse and human PPARδ. The anti-inflammatory potential of human PPARδ was also similar to mouse PPARδ in liver and isolated macrophages. CONCLUSIONS AND IMPLICATIONS: These data indicate that human PPARδ can compensate for mouse PPARδ in the regulation of lipid metabolism and inflammation. Overall, this novel PPARδ KI mouse model shows full responsiveness to pharmacological challenge and represents a useful tool for the preclinical assessment of PPARδ activators with species-specific activity.

Exposure of red bone marrow to ionising radiation from natural and medical sources in France.

This paper presents a review of available data on the average level of equivalent dose to the red bone marrow in France, due to natural and some medical exposures. The sources of exposure to ionising radiation which were studied are medical examinations involving x-rays (diagnostic radiology), inhalation of radon and thoron, cosmic irradiation, terrestrial irradiation and intakes of natural radionuclides. This review is based, where possible, on specific data for France. The influence of age on exposure levels is studied when data are available. The total equivalent dose to the red bone marrow is estimated at 2.4 mSv year(-1), 2.7 mSv year(-1) and 5.4 mSv year(-1) respectively for adults (> or =18 years old), children (3-17 years old) and infants (0-1 year old). The high level of exposure for infants is explained by the important contribution of 210Po. The average equivalent dose to the red bone marrow for adults is also compared with the average committed effective dose due to the same exposure sources. The importance of medical exposure and natural exposure due to intake of natural radionuclides emphasises the importance of obtaining more information about these sources of exposure.

Fungal pathogen protection in potato by expression of a plant defensin peptide.

Defensins are small cysteine-rich peptides with antimicrobial activity. We demonstrate that the alfalfa antifungal peptide (alfAFP) defensin isolated from seeds of Medicago sativa displays strong activity against the agronomically important fungal pathogen Verticillium dahliae. Expression of the alfAFP peptide in transgenic potato plants provides robust resistance in the greenhouse. Importantly, this resistance is maintained under field conditions. There have been no previous demonstrations of a single transgene imparting a disease resistance phenotype that is at least equivalent to those achieved through current practices using fumigants.

[Assessment of the risk of radiation-induced leukaemia in the vicinity of nuclear installations: the Nord-Cotentin radio-ecological study]. / Evaluation du risque de leucémie radio-induite à proximité d'installations nucléaires: l'étude radio-écologique Nord-Cotentin.

BACKGROUND: A radioecological study has been launched in 1997 to perform a realistic assessment of radiation doses received by the population aged 0 to 24 years who lived in the vicinity of the La-Hague nuclear reprocessing plant (Nord-Cotentin, France), and to estimate the associated risk of leukaemia between 1978 and 1996. METHODS: The Working Group in charge of the study included representatives of French radiation protection institutes, nuclear operators, members of environmental organisations and international experts. The methodology retained developed three steps RESULTS: The reconstructed cohort included 6656 individuals born between 1954 and 1996, who lived in the study area for at least one year between 1978 and 1996 before the age of 25. The number of person-years between 1978 and 1996 is 69 308. On the basis of the calculated doses, the estimated number of radiation-induced leukaemia attributable to releases from local nuclear installations was less than 0.002 for the period 1978-1996. CONCLUSIONS: This result constitutes a best estimate of the risk of radiation-induced leukaemia among young people in the canton of Beaumont-Hague. Nevertheless, this estimation must be interpreted in the light of the limits inherent to the risk assessment process and some participants of the Working Group expressed some reservations. The estimated number of radiation-induced leukaemia attributable to local nuclear installations is low in comparison to the 4 cases of leukaemia observed by epidemiological studies during the same period. It is therefore very unlikely that releases from local nuclear installations could notably explain the high incidence of leukaemia observed among young people in the canton of Beaumont-Hague.

Methodology and results of the Nord-Cotentin radioecological study.

Epidemiological studies have shown a trend towards an excess number of leukaemia cases in the region of Nord-Cotentin (France) where, in particular, the La Hague nuclear reprocessing plant is located. In 1997, it was suggested that the risk of leukaemia was associated with some aspects of lifestyle, in particular, the consumption of local seafood and use of local beaches. To respond to public concern, the French Ministries of the Environment and Health decided to commission complementary epidemiological studies and a detailed radioecological analysis. The radioecological study was entrusted to a group of experts with various backgrounds (inspectors, governmental experts, operators, experts from non-governmental laboratories and foreign experts)--the Nord-Cotentin Radioecology Group. Its principal objective was to assess realistically the exposure to ionising radiation of young people from 0 to 24 years of age who had lived near the La Hague nuclear reprocessing plant and to estimate their risk of radiation-induced leukaemia from 1978 through 1996, the period covered by the epidemiological studies. The Group chose to use a three-stage approach: reconstruction of the population of young people from 0 to 24 years who resided in the region between 1978 and 1996, assessment of their exposure to all sources of ionising radiation, and estimation of the risk of radiation-induced leukaemia attributable to this exposure. The collective red bone marrow dose due to the discharges from the local nuclear facilities from Nord-Cotentin has thus been estimated at approximately 0.5 man-Sv, which is less than 0.2% of the total exposure to ionising radiation, including natural and medical sources and fallout from atmospheric testing and the accident at Chernobyl. The number of cases of radiation-induced leukaemia attributable to discharges from the local nuclear facilities based on the estimated level of exposure was around 0.002 over this period. This is the best estimate, in the current state of knowledge, of the incidence of radiation-induced leukaemia attributable to environmental exposure to ionising radiation among the young people living in the vicinity of the La Hague reprocessing plant based on mean habits for the population. This estimate must be interpreted in the light of the limitations inherent in the risk assessment process, and some participants in the Nord-Cotentin Radioecology Group have expressed reservations about it. Nonetheless, the number of cases estimated here is low in comparison to the four cases of leukaemia observed during the same period. It is thus very improbable that exposure attributable to local nuclear facilities is implicated to any salient degree in the elevated incidence of leukaemia observed in this region among young people.

Exploiting the full potential of disease-resistance genes for agricultural use.

Effective and sustained control of fungal pathogens and nematodes is an important issue for all agricultural systems. Global losses caused by pathogens are estimated to be 12% of the potential crop production [1], despite the continued release of new resistant cultivars and pesticides. Furthermore, fungi are continually becoming resistant to existing resistance genes and fungicides, and a few of the pesticides are being withdrawn from the market for environmental reasons. In addition to reducing crop yield, fungal diseases often lower crop quality by producing toxins that affect humans and human health. Additional methods of disease control are therefore highly desirable. Breeding programs based on plant disease-resistance genes are being optimized by incorporating molecular marker techniques and biotechnology. These efforts can be expected to result in the first launches of new disease-resistant crops within the next five years.

Synthesis of hydrazinopeptides using solid-phase N-electrophilic amination: extension to the Fmoc/tert-butyl strategy and chemistry of the N-N bond in strong acid media.

The synthesis of hydrazinopeptides using solid-phase N-electrophilic amination was extended to the Fmoc/tert-butyl strategy. Both Boc/benzyl and Fmoc/tert-butyl strategies led to the isolation of by-products arising from the partial instability of the N-N bond during the final cleavage and deprotection step. Two paths of decomposition have been shown: the cleavage of the N-N bond leading to the regeneration of the amine and a Hofmann-type elimination yielding original dianisyl adducts. Our data suggest that the Fmoc/tert-butyl strategy is better suited for the synthesis of hydrazinopeptides.

Direct evidence of cytoplasmic delivery of PKC-alpha, -epsilon and -zeta pseudosubstrate lipopeptides: study of their implication in the induction of apoptosis.

Protein kinases C (PKC) are serine/threonine kinase enzymes involved in the mechanism of cell survival. Their pseudosubstrate sequences are autoinhibitory domains, which maintain the enzyme in an inactive state in the absence of allosteric activators, thus representing an attractive tool for the modulation of different PKC isoforms. Here, we report the use of palmitoylated modified PKC-alpha, -epsilon, and -zeta pseudosubstrate peptides, and determine their intracellular distribution together with their respective PKC isoenzymes. Finally, we propose that the differential distribution of the peptides is correlated with a selective induction of apoptosis and therefore argues for different involvement of PKC isoforms in the anti-apoptotic program.

Reactivity of Lys(NH2)-containing peptides toward endopeptidases.

Lys(NH2)-containing peptides were subjected to various proteolytic enzymes which were selected for their well-documented specificity for arginyl and/or lysyl peptide bonds. Lys(NH2)-containing peptides were cleaved more rapidly by clostripain than the corresponding lysyl peptides. On the other hand, they proved to be resistant to Achromobacter protease I hydrolysis. The modified peptides synthesized in this study were more stable than the arginyl and lysyl analogues when incubated with trypsin or thrombin. The same tendency was observed when Lys(NH2)-containing peptides were incubated in diluted human serum, suggesting that the replacement of Arg or Lys by Lys(NH2) could be used to increase the stability of peptides in vivo.

Type 1 CD4(+) T-cell help is required for induction of antipeptide multispecific cytotoxic T lymphocytes by a lipopeptidic vaccine in rhesus macaques.

We have optimized the induction of antiviral cytotoxic T lymphocytes (CTL) in rhesus macaques by a lipopeptide vaccine containing seven peptides from simian immunodeficiency virus (SIV) Nef and Gag proteins and a strong T-helper peptide from tetanus toxoid (TT) that is promiscuous in humans (peptide TT 830-846). Two of the eight immunized macaques showed T-helper (Th) cell proliferation and a specific synthesis of gamma interferon in response to TT 830-846 peptide. They also showed multispecific cytotoxic activity against three to five of the immunizing SIV peptides. These results show the importance of a strong specific type 1 Th response for inducing a multispecific CTL response in vivo, which is essential for the development of an anti-human immunodeficiency virus vaccine.

Synthesis of lipopeptides using hydrazone chemical ligation.

In this paper we report that a hydrazinopeptide synthesized using solid-phase N-electrophilic amination with N-Boc-3-(4-cyanophenyl)oxaziridine reacted with a lipophilic peptide aldehyde to give the corresponding hydrazone plus an unexpected 1,3,4-oxadiazolidinopeptide containing two peptide aldehyde units. This methodology allows the synthesis of large lipopeptides.

Identification of a sequence-dependent reversible acylation of tosylarginine in a peptidyl-resin reacted with isonicotinyl p-nitrophenylcarbonate.

In this paper, we report on the identification of an unexpected acylation that occurred on the solid phase when a peptide containing an unprotected lysyl and a tosyl-protected arginyl residue was treated with a large excess of isonicotinyl p-nitrophenylcarbonate. NMR and matrix-assisted laser desorption/ionisation -post-source decay analysis of the purified peptide demonstrated the presence of one extra isonicotinyloxycarbonyl (iNoc) group located on the omega nitrogen atom of the arginine which was adjacent to the Lys(iNoc). The desired peptide was obtained by quantitative removal of the unwanted iNoc group during a brief treatment with diluted aqueous hydrazine.

Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin.

The lesion-mimic mutants of certain plants display necrotic lesions resembling those of the hypersensitive response and activate local and systemic defense responses in the absence of pathogens. We have engineered a lesion-mimic phenotype in transgenic Russet Burbank potato plants through constitutive expression of a bacterio-opsin (bO) proton pump derived from Halobacterium halobium. Transgenic potato plants exhibiting a lesion-mimic phenotype had increased levels of salicylic acid and overexpressed several pathogenesis-related messenger RNAs, all hallmarks of systemic acquired resistance (SAR). The lesion-mimic plants also displayed enhanced resistance to the US1 isolate (A1 mating type) of a fungal pathogen, Phytophthora infestans, a causal agent of late blight disease. In contrast, little resistance was observed against the US8 isolate (A2 mating type) of this pathogen. Furthermore, a majority of the transgenic plants displaying the lesion-mimic phenotype had increased susceptibility to potato virus X. The tubers of these plants were not resistant to the bacterial pathogen Erwinia carotovora. These results indicate that expression of bO can result in the activation of defense responses in transgenic potato plants and show for the first time that bO expression can confer resistance to a pathogenic fungus. However, our results also demonstrate that like SAR, this "engineered" resistance is likely to be limited to certain pathogens and particular cultivars.

Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster.

In tomato, resistance to Pseudomonas syringae pv. tomato (Pst) strains expressing the avirulence gene avrPto requires the presence of at least two host genes, designated Pto and Prf. Here we report that Prf encodes a protein with leucine-zipper, nucleotide-binding, and leucine-rich repeat motifs, as are found in a number of resistance gene products from other plants. prf mutant alleles (4) were found to carry alterations within the Prf coding sequence. A genomic fragment containing Prf complemented a prf mutant tomato line both for resistance to Pst strains expressing avrPto and for sensitivity to the insecticide Fenthion. Prf resides in the middle of the Pto gene cluster, 24 kb from the Pto gene and 500 bp from the Fen gene.

Intergeneric transfer and functional expression of the tomato disease resistance gene Pto.

Plant disease resistance loci have been used successfully in breeding programs to transfer traits from resistant germplasm to susceptible plant cultivars. The molecular cloning of plant disease resistance genes now permits the transfer of such traits across species boundaries by genetic transformation of recipient hosts. The tomato disease resistance gene Pto confers resistance to strains of the bacterial pathogen Pseudomonas syringae pv tomato expressing the avirulence gene avrPto. Transformation of Nicotiana benthamiana with Pto results in specific resistance to P. s. pv tabaci strains carrying avrPto. The resistant phenotype is manifested by a strong inhibition of bacterial growth and the ability to exhibit a hypersensitive response. Resistance cosegregates with the Pto gene in transgene selfings and testcrosses. Our results demonstrate the conservation of disease resistance functions across genus boundaries and suggest that the utility of host-specific resistance genes can be extended by intergeneric transfer.

Use of a gene expression system based on potato virus X to rapidly identify and characterize a tomato Pto homolog that controls fenthion sensitivity.

A novel transient gene expression system was used to study both the tomato disease resistance gene Pto and a Pto homolog designated Fen. The gene expression system was based on potato virus X (PVX). Tomato plants that were both susceptible to strains of Pseudomonas syringae pv tomato carrying the corresponding avirulence gene avrPto and insensitive to the insecticide fenthion were infected with in vitro-generated transcripts of PVX derivatives containing either Pto or Fen. Expression of the Pto gene from the virus genome failed to elicit P.s. tomato resistance, indicating that the PVX system is not suitable for the study of Pto. However, expression of the Fen gene resulted in sensitivity to fenthion. The utility of the PVX gene expression system was further demonstrated through structure/function studies of the Fen gene. A correlation was shown between Fen protein kinase activity and the ability of this protein to confer fenthion sensitivity to tomato. Furthermore, it was demonstrated that mutation of a putative N-terminal myristoylation site, proposed to be involved in membrane targeting, rendered the Fen protein inactive. Analysis of a Pto-Fen chimeric gene allowed the fenthion sensitivity domain to be localized to the C-terminal part of the Fen protein. Interestingly, expression of the Fen kinase from the PVX genome in Nicotiana spp resulted in a fenthion-independent necrotic response. Our results support the involvement of the Fen gene in a signal transduction pathway(s).

Differential repair of excision gaps generated by transposable elements of the 'Ac family'.

Studies on transposable elements of the Ac family have led to different models for excision gap repair in either plants or Drosophila. Excision products generated by the plant transposable elements Ac and Tam3 imply a more or less straightforward ligation of broken ends; excision products of the Drosophila P element indicate the involvement of 'double-strand break' (DSB) repair. Recent findings that excision products of Ac and Tam3 can also contain traces of the element ends indicate, however, that DSB repair might be an alternative repair mechanism in plants. A functional DSB repair mechanism in plants can also be deduced from the observed rapid increases of Ac copy number during plant development and from the involvement of Ac in the generation of internal Ac deletions. On the other hand, alternative repair mechanisms may also be functional in Drosophila, because some of the 'footprints' generated upon P excision can be explained by a mechanism that has been postulated for excision gap repair in plants. It is concluded that plants and Drosophila can use similar repair mechanisms, but that the predominance of a certain repair mechanism is determined by the host.

Transposition pattern of a modified Ds element in tomato.

Several aspects of transposition of an in vitro modified Ds element are described. This Ds element, designated Ds-r, is equipped with bacterial plasmid sequences and can, therefore, be rescued from the plant genome. Our results indicate that the Ds-r element has a 'late' timing of transposition from T-DNAs. This feature of the element might be advantageous for tagging experiments because it leads to independently transposed germinally transmitted elements. Furthermore, it is shown that Ds-r transposition generates clusters of insertions, indicating that 'genes to be tagged' should be located in genomic regions covered by insertions.

Supported PCR: an efficient procedure to amplify sequences flanking a known DNA segment.

We describe a novel modification of the polymerase chain reaction for efficient in vitro amplification of genomic DNA sequences flanking short stretches of known sequence. The technique utilizes a target enrichment step, based on the selective isolation of biotinylated fragments from the bulk of genomic DNA on streptavidin-containing support. Subsequently, following ligation with a second universal linker primer, the selected fragments can be amplified to amounts suitable for further molecular studies. The procedure has been applied to recover T-DNA flanking sequences in transgenic tomato plants which could subsequently be used to assign the positions of T-DNA to the molecular map of tomato. The method called supported PCR (sPCR) is a simple and efficient alternative to techniques used in the isolation of specific sequences flanking a known DNA segment.