Production of pharmaceutical proteins by transgenic animals.

Proteins are involved in a majority of the biochemical events that take place in all living organisms. Protein synthesis is directed by genes. All genes contain two major DNA regions. The region containing the genetic message proper (the 'coding region') is preceded by a regulatory region ('the promoter'), which determines when and in which organs a given gene must produce the corresponding protein. The techniques of genetic engineering allow the association of the coding region from one gene with the regulatory region from another gene. The expression of these recombinant genes may be achieved in cultured cells, in transgenic animals or in plants. This leads to the production of the corresponding proteins, including pharmaceutical proteins. Milk from transgenic animals is one potential source of pharmaceutical proteins. To achieve this, the promoters from milk protein genes are bound to DNA fragments containing the coding region of the genes of interest. The desired proteins are then taken from the milk and purified. Two human pharmaceutical proteins are on the market and about 20 projects are in development. One of the proteins produced in milk, antithrombin III, is an anticoagulant and the other, human C1-esterase inhibitor, is an anti-inflammatory. Several human proteins have been produced in the egg white of transgenic chickens and one has been approved by the United States Federal Drug Administration. This process has also been used to modify antibodies in cows. The genes that code for antibodies in the cow were deleted and replaced by human antibody genes. These cows, immunised by various antigens, then secreted purely human antibodies in their blood. Antibodies from such cows were able to attenuate the effects of Ebola virus in human patients.

Les protéines sont des acteurs moléculaires majeurs des organismes vivants. Leur synthèse résulte du décodage des gènes. Essentiellement, tous les gènes contiennent deux régions d'ADN majeures. La région contenant le message génétique proprement dit (région codante) est précédée d'une région de régulation (le promoteur) qui détermine quand et dans quels organes un gène donné doit produire la protéine correspondante. Les techniques de génie génétique permettent l'association de la région codante d'un gène donné à une région régulatrice d'un autre gène. L'expression des gènes recombinants peut être réalisée dans des cellules cultivées, dans des animaux ou des plantes transgéniques. Ceci conduit à la production des protéines correspondantes comprenant des protéines pharmaceutiques. Le lait d'animaux transgéniques a été retenu comme l'une des sources potentielles de protéines pharmaceutiques. Pour atteindre cet objectif, les promoteurs des gènes des protéines du lait sont liés à des fragments d'ADN contenant les gènes d'intérêt. Les protéines sont ainsi retrouvées dans le lait et purifiées. Deux protéines pharmaceutiques humaines sont sur le marché et une vingtaine de projets sont en cours de développement. L'une des protéines produites dans le lait, l'antithrombine III, est un anticoagulant, tandis que l'autre, l'inhibiteur de la C1-estérase humaine, est un anti-inflammatoire. Plusieurs protéines humaines ont été produites dans du blanc d'oeuf de poulet transgénique et l'une d'entre elles a été approuvée par l'Agence américaine des produits alimentaires et médicamenteux. Cette méthode a également été utilisée pour modifier l'expression génétique des anticorps chez des vaches. Des vaches dont les gènes codant les anticorps ont été inactivés et remplacés par les gènes d'anticorps humains ont été obtenues. Ces vaches immunisées par divers antigènes sécrètent dans leur sang des anticorps purement humains. Certains anticorps de telles vaches ont été capables d'atténuer les effets du virus Ebola chez les patients.

Las proteínas participan en la mayoría de las reacciones bioquímicas que tienen lugar en todos los organismos vivos. La síntesis de proteínas está regida por los genes. Todos los genes contienen dos regiones principales de ADN. La región que contiene el mensaje genético propiamente dicho («región codificante¼) viene precedida de una región reguladora («promotora¼), que determina cuándo y en qué órganos un determinado gen debe expresar la correspondiente proteína. Las técnicas de ingeniería genética permiten asociar entre sí la región codificante de un gen y la región reguladora de otro gen. Estos genes recombinantes pueden ser llevados a expresarse en cultivos celulares, animales transgénicos o plantas, lo que resulta en la producción de las correspondientes proteínas, entre ellas proteínas farmacéuticas. La leche de animales transgénicos es una posible fuente de proteínas farmacéuticas. Para obtenerlas se unen las secuencias promotoras de los genes de proteínas lácteas con fragmentos de ADN que contienen la región codificante de los genes que interesan. Después se extraen de la leche y se purifican las proteínas deseadas. Ahora mismo hay dos proteínas farmacéuticas humanas que ya están comercializadas, además de unos 20 proyectos en curso. Una de las proteínas fabricadas en leche, la antitrombina III, es un anticoagulante, y la otra, inhibidora de la C1 esterasa humana, un antiinflamatorio. Varias proteínas humanas, una de ellas ya aprobada por la Administración de Alimentos y Medicamentos de los Estados Unidos, han sido producidas en la clara de huevo de gallinas transgénicas. Este proceso también ha sido utilizado para modificar anticuerpos en vacas. Los genes que codifican los anticuerpos de la vaca son suprimidos y sustituidos por genes de anticuerpos humanos. Estas vacas, expuestas a varios antígenos, secretan y envían al torrente sanguíneo anticuerpos humanos puros. Con los anticuerpos obtenidos a partir de tales vacas se pudieron atenuar los efectos del virus Ebola en pacientes humanos.

Genetically modified animals from life-science, socio-economic and ethical perspectives: examining issues in an EU policy context.

The interdisciplinary EC consortium (the PEGASUS project) aimed to examine the issues raised by the development, implementation and commercialisation of genetically modified (GM) animals, and derivative foods and pharmaceutical products. The results integrated existing social (including existing public perception) environmental and economic knowledge regarding GM animals to formulate policy recommendations relevant to new developments and applications. The use of GM in farmed animals (aquatic, terrestrial and pharmaceutical) was mapped and reviewed. A foresight exercise was conducted to identity future developments. Three case studies (aquatic, terrestrial and pharmaceutical) were applied to identify the issues raised, including the potential risks and benefits of GM animals from the perspectives of the production chain (economics and agri-food sector) and the life sciences (human and animal health, environmental impact, animal welfare and sustainable production). Ethical and policy concerns were examined through application of combined ethical matrix method and policy workshops. The case studies were also used to demonstrate the utility of public engagement in the policy process. The results suggest that public perceptions, ethical issues, the competitiveness of EU animal production and risk-benefit assessments that consider human and animal health, environmental impact and sustainable production need to be considered in EU policy development. Few issues were raised with application in the pharmaceutical sector, assuming ethical and economic issues were addressed in policy, but the introduction of agricultural GM animal applications should be considered on a case-by-case basis.

Rabbit milk protein genes: from mRNA identification to chromatin structure.

Milk protein genes are among the most intensively expressed and they are active only in epithelial mammary cells of lactating animals. They code for proteins which represent 30% of the proteins consumed by humans in developed countries. Mammary gland development occurs essentially during each pregnancy. This offers experimenters attractive models to study the expression mechanisms of genes controlled by known hormones and factors (prolactin, glucocorticoids, progesterone, insulin-like growth factor-1 and others) as well as extracellular matrix. In the mid-1970s, it became possible to identify and quantify mRNAs from higher living organisms using translation in reticulocyte lysate. A few years later, the use of radioactive cDNAs as probes made it possible for the quantification of mRNA in various physiological situations using hybridisation in the liquid phase. Gene cloning offered additional tools to measure milk protein mRNAs and also to identify transcription factors. Gene transfer in cultured mammary cells and in animals contributed greatly to these studies. It is now well established that most if not all genes of higher eukaryotes are under the control of multiple distal regulatory elements and that local modifications of the chromatin structure play an essential role in the mechanisms of differentiation from embryos to adults. The technique, known as ChIP (chromatin immunoprecipitation), is being implemented to identify the factors that modify chromatin structure at the milk protein gene level during embryo development, mammogenesis and lactogenesis, including the action of hormones and extracellular matrix. Transgenesis is not just a tool to study gene regulation and function, it is also currently used for various biotechnological applications including the preparation of pharmaceutical proteins in milk. This implies the design of efficient vectors capable of directing the secretion of recombinant proteins in milk at a high concentration. Milk protein gene promoters and long genomic-DNA fragments containing essentially all the regulatory elements of milk protein genes are used to optimise recombinant protein production in milk.

[Confinement and consumption of cloned and transgenic animals]. / Confinement et consommation des animaux clonés et transgéniques.

Reproduction by cloning can eliminate some of the problems inherent to sexual reproduction, but it creates others. The genetic heritage of nucleus donor cells and the genetic status of clones are not precisely known. Furthermore, reprogramming of the genome of nucleus donor cells by the ovocyte cytoplasm is often incomplete. Animals obtained through cloning are thus essentially genetically identical to their genitors, but they are often epigenetically modified, with unpredictable effects. Transgenesis results in most cases from the addition to a genome of one or more known genes. The direct and indirect effects of transgenesis cannot all be predicted. Specific confinement measures make it possible to raise animals in high-security conditions, preventing their dissemination in the human food chain, in animal feed or in the environment. The toxicity, allergenicity and infectiousness of cloned ortransgenic animals can be evaluated by means of tests.

Use of transgenic animals to improve human health and animal production.

Contents Transgenic animals are more widely used for various purposes. Applications of animal transgenesis may be divided into three major categories: (i) to obtain information on gene function and regulation as well as on human diseases, (ii) to obtain high value products (recombinant pharmaceutical proteins and xeno-organs for humans) to be used for human therapy, and (iii) to improve animal products for human consumption. All these applications are directly or not related to human health. Animal transgenesis started in 1980. Important improvement of the methods has been made and are still being achieved to reduce cost as well as killing of animals and to improve the relevance of the models. This includes gene transfer and design of reliable vectors for transgene expression. This review describes the state of the art of animal transgenesis from a technical point of view. It also reports some of the applications in the medical field based on the use of transgenic animal models. The advance in the generation of pigs to be used as the source of organs for patients and in the preparation of pharmaceutical proteins from milk and other possible biological fluids from transgenic animals is described. The projects in course aiming at improving animal production by transgenesis are also depicted. Some the specific biosafety and bioethical problems raised by the different applications of transgenesis, including consumption of transgenic animal products are discussed.

[Impact of genetic modifications on infectious diseases]. / L'impact des modifications génétiques sur les maladies infectieuses.

Genetic engineering offers the theoretical possibility to transfer any natural or modified gene into any living organism. This generates new and diverse situations which may contribute to the spreading of infectious diseases or on the contrary to control them. Problems may theoretically come from uncontrolled genes providing resistance to antibiotics, from the activation of genomic retroviral sequences, from enhanced sensitivity of the organism to pathogens, as well as from the generation of mutated microorganisms with a higher pathogenecity. On the contrary, various genetic modifications may create organisms resistant to infectious diseases, generate safe and efficient recombinant vaccines, or provide patients with proteins which stimulate their defense mechanisms. The major impacts of genetic modifications in the development of infectious diseases or on the contrary in their eradication are analyzed in this article.

Hypodermin A, a new inhibitor of human complement for the prevention of xenogeneic hyperacute rejection.

BACKGROUND: Hyperacute rejection (HAR) of discordant xenografts in the pig-to-human combination can be prevented using tranplants expressing transgenic molecules that inhibit human complement. Hypodermin A (HA), a serine esterase that degrades C3, was tested in the guinea-pig-to-rat and in the pig-to-human combinations. METHODS: Hypodermin A was tested in vitro, ex vivo, and in vivo models of HAR in the guinea-pig-to-rat combination. Hamster ovary cells (CHO) and a line of porcine aortic endothelial cells (PAEC11) were transfected with HA complementary DNA (cDNA). RESULTS: The pattern of degradation of rat and human C3 by HA was different (multiple bands lower than 40 kDa) from the physiologic pattern observed after spontaneous degradation of rat C3 or physiologic activation of human C3. The CH50 activity in serum was significantly lower in rats treated with 3.2 mg HA/kg than in untreated rats (45 +/- 16 U/ml vs. 700 +/- 63 U/ml, P < 0.05). Sera from rats injected with 3.2 mg/kg of HA were less effective in lysing guinea-pig endothelial cells (12 +/- 7%) than normal rat sera (79 +/- 3%; P < 0.001). Ex vivo, guinea-pig hearts perfused by rat serum supplemented with HA survived longer than those perfused by non-treated serum (210 +/- 34 and 154 +/- 71 min, respectively; P < 0.05). In vivo, guinea-pig hearts transplanted into HA treated rats survived longer than in non-treated rats (27 +/- 5 min vs. 13 +/- 4 min; P < 0.001). In the presence of human serum, smaller amounts of C6 and C5b-9 were deposited onto HA-transfected CHO cells than onto control cells. The mHA-PAEC11 cells were significantly more resistant to lysis by human C than control PAEC11 cells. CONCLUSIONS: These data suggest that transgenic HA could be used to prevent hyperacute xenogeneic rejection.

Extracellular matrix regulates alpha s1-casein gene expression in rabbit primary mammary cells and CCAAT enhancer binding protein (C/EBP) binding activity.

Previous studies have shown that both the signal transducer and activator of transcription 5 (STAT5) and the CCAAT enhancer binding proteins (C/EBPs) are involved in the regulation of casein gene expression by mammary epithelial cells. Prolactin (Prl) activation of STAT5 is necessary for casein gene expression. The extracellular matrix (ECM) regulates also casein gene expression. Here, we have investigated whether ECM regulates C/EBPs activity in primary rabbit mammary epithelial cells. Isolated primary mammary cells were cultured on plastic or on floating collagen I gel. Prolactin induced alphas 1-casein gene expression when cells were cultured on collagen but not on plastic. It is noteworthy that activated STAT5 was detected in both culture conditions. Several STAT5 isoforms (STAT5a, STAT5b, and other STAT5 related isoforms, some with lower molecular weight than the full-length STAT5a and STAT5b) were detected under the different culture conditions. However, their presence was not related to the expression of alphas 1-casein gene. The binding of nuclear factors to a C/EBP specific binding site and the protein level of C/EBPbeta differed in cells cultured on plastic or on collagen but these parameters were not modified by Prl. This suggests that C/EBP binding activity was regulated by ECM and not by Prl. Interestingly, these modifications were correlated to the expression of the alphas 1-casein gene. Hence, the activation of the alphas 1-casein gene expression depends on two independent signals, one delivered by Prl via the activation of STAT5, the other delivered by ECM via C/EBP.

Expression of a single betaalpha chain protein of equine LH/CG in milk of transgenic rabbits and its biological activity.

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked alpha- and beta-subunits. eCG possesses the particularity to bind to both LH and FSH receptors in species other than horses and to have a prolonged plasma half-life. All these properties make it of utmost interest for livestock fertilization program. Up to now, the only source of eCG is the serum of pregnant mare. Rabbit mammary gland is considered as a system able to produce recombinant glycoproteins in sufficient quantity for pharmaceutical use. Here we described the production of a recombinant single betaalpha chain of eLH/CG in the milk of transgenic rabbit. The construction of a single-chain permits to by-pass the problem of association-dissociation of the subunits. This recombinant hormone is greatly expressed (21.7 mg/l) and presents similar in vitro LH and FSH bioactivities. However, betaalphaeLH/CG shows an extremely rapid clearance (approximately 10 min), which could explain the absence of in vivo biological activity. So the rabbit mammary gland is not appropriate for the production of a recombinant active eLH/CG.

Transgenic animal bioreactors.

The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk, egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant proteins at an industrial scale. Although the first recombinant protein produced by transgenic animals is expected to be in the market in 2000, a certain number of technical problems remain to be solved before the various systems are optimized. Although the generation of transgenic farm animals has become recently easier mainly with the technique of animal cloning using transfected somatic cells as nuclear donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried out for the last 15 years have shown that the expression of the transgene is predictable only to a limited extent. This is clearly due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes from the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result sometime in poorly functional expression vectors. One possibility consists in using long genomic DNA fragments contained in YAC or BAC vectors. The other relies on the identification of the major important elements required to obtain a satisfactory transgene expression. These elements include essentially gene insulators, chromatin openers, matrix attached regions, enhancers and introns. A certain number of proteins having complex structures (formed by several subunits, being glycosylated, cleaved, carboxylated...) have been obtained at levels sufficient for an industrial exploitation. In other cases, the mammary cellular machinery seems insufficient to promote all the post-translational modifications. The addition of genes coding for enzymes involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate some mammary infectious diseases.

The efficiency of different IRESs (internal ribosomes entry site) in monocistronic mRNAS.

The IRES from poliovirus and from encephalomyocarditis virus (EMCV) added between the cap and the AUG initiator codon were strong inhibitors of chloramphenicol acetyltransferase gene expression in three different cell types. The poliovirus IRES also inhibited bGH (bovine growth hormone) cDNA expression in the HC11 mammary cell line when added between the rabbit whey acidic gene promoter and the cDNA whereas the HTLV-1 IRES showed a stimulatory effect in the same situation. RNA stem loops were added before HTLV-1 (SUR) and the BiP (Immunoglobulin heavy-chain Binding Protein) IRESs followed by the firefly luciferase gene under the control of Rous sarcoma virus (RSV) promoter. The RNA loops abolished the expression of the reporter gene almost completely. These data suggest that the different IRESs may favour or inhibit translation of monocistronic mRNA.

[Impact of transgenes and cloning on xenografts]. / L'impact de la transgen¿ese et du clonage sur la x¿enogreffe.

Transgenesis can theoretically add a foreign gene or specifically replace an endogenous gene by another gene. Gene addition in mammals is generally achieved by DNA microinjection into one-cell embryos. Gene replacement implies homologous recombination in cultured cells which must be selected and remain capable of generating a living organism. The use of totipotent cells can, though currently in the mouse only, lead to the generation of chimeric animals transmitting the genetic modification to offsprings. The embryo cloning technique has recently allowed the use of somatic fetal cells, in which gene replacement occurred to generate living sheeps. This technique is being extended to other domestic ruminants, to pigs and rabbits. The mouse, rat and rabbit are being used as models to define the genes which should be added or inactivated to reduce rejections of xenografts. Transgenic pigs harbouring the human CD59 or the DAF genes have been obtained by several groups. Heart, kidney and isolated cells from these animals are more resistant to hyperacute rejection when grafted to experimental primates. Additional genes are to be added in the future to inhibit the other rejection mechanisms as soon as their action has been demonstrated in laboratory animals. Experiments in progress aim at inactivating the gene for Gal: 1-3-galactosyl transferase by homologous recombination in the pig genome. Transgenesis might also be used to prevent expression of endogenous pig retroviral vectors and to prepare recombinant proteins having antirejection activity from the milk of animals.

The stimulation of gene expression by the R region from HTLV-1 and BLV.

The 5' untranslated regions (5'UTR) of mRNA are known to stimulate or inhibit more or less translation. SR alpha, an association of SV40 early gene promoter and of the R region plus the first 39 nucleotides of the U5 region (designated as R) from the human T-cell leukemia virus (HTLV-1) is currently used to stimulate expression of various coding regions. Its effect is considered to take place at the translational level. In all studies published so far, the R region was associated with the promoter and 5'UTR from SV40 early genes. In the present work, the role of SV40 5'UTR and HTLV-1R region was evaluated separately using different promoters, reporter genes and cells. Both SV40 5'UTR (SU) and R region (R) from HTLV-1 stimulated separately the expression of adjacent reporter genes. When associated, the SV40 5'UTR and the R region from HTLV-1 (SUR) were a more potent stimulator of gene expression and their effects were more than additive. This effect was very potent in HeLa and HC11 cells and almost inexistent in CHO and COS 7 cells. It was of various intensity in other cell types including bird and fish cells. The presence of SUR in gene constructs favoured the accumulation of the mRNAs. SUR stimulated gene expression when added between the cap and the initiation codon. Unexpectedly, SUR was never inhibitory. SUR can therefore be considered essentially as potent and specific stimulator of gene expression favoring mRNA accumulation.

The optimal use of IRES (internal ribosome entry site) in expression vectors.

In higher eucaryotes, natural bicistronic mRNA have been rarely found so far. The second cistron of constructed bicistronic mRNAs is generally considered as not translated unless special sequences named internal ribosome entry site (IRES) are added between the two cistrons. These sequences are believed to recruit ribosomes independently of a cap structure. In the present report, a new IRES found in the HTLV-1 genome is described. A systematic study revealed that this IRES, but also the poliovirus (polio) and the encephalomyocarditis virus (EMCV) IRES work optimally when they are added about 100 nucleotides after the termination codon of the first cistron. Unexpectedly, these IRES became totally inefficient when added after 300-500 nucleotide spacers. This result and others are not compatible with the admitted mechanism of IRES action. The IRES appear to be rather potent translation stimulators. Their effects are particularly emphasized in cells in which the normal mechanism of translation initiation is inhibited. For these reasons, we suggest to call IRES rescue translation stimulators (RTS).

Association of the 5'HS4 sequence of the chicken beta-globin locus control region with human EF1 alpha gene promoter induces ubiquitous and high expression of human CD55 and CD59 cDNAs in transgenic rabbits.

Whatever its field of application, animal transgenesis aims at a high level of reproducible and stable transgene expression. In the case of xenotransplantation, prevention of hyperacute rejection of grafts of animal origin requires the use of organs expressing human inhibitors of complement activation such as CD55 (DAF) and CD59. Pigs transgenic for these molecules have been produced, but with low and variable levels of expression. In order to improve cDNA expression, a vector containing the 5'HS4 region from the LCR of the chicken beta-globin locus and the promoter and the first intron from the human EF1 alpha gene, was used to co-express human CD55 and CD59 cDNAs in transgenic rabbits. The transgenic lines with the 5'HS4 region displayed dramatically enhanced CD55 and CD59 mRNA concentrations in brain, heart, kidney, liver, lung, muscle, spleen and aortic endothelial cells in comparison with the transgenic lines without the 5'HS4 region. In the absence of the 5'HS4 region, only some of the transgenic lines displayed specific mRNAs and at low levels. Human CD55 and CD59 proteins were detectable in mononuclear cells from transgenic rabbits although at a lower level than in human mononuclear cells. On the other hand, primary aortic endothelial cells from a bi-transgenic line were very efficiently protected in vitro against human complement-dependent lysis. Transgenic rabbits harbouring the two human inhibitors of complement activation, CD55 and CD59, can therefore be used as new models in xenotransplantation. Moreover, the vector containing the 5'HS4 region from the LCR of the chicken beta-globin locus seems appropriate not only for xenotransplantation but also for any other studies involving transgenic animals in which cDNAs have to be expressed at a high level in all cell types.

Internal ribosome entry sites (IRESs): reality and use.

IRESs are known to recruit ribosomes directly, without a previous scanning of untranslated region of mRNA by the ribosomes. IRESs have been found in a number of viral and cellular mRNAs. Experimentally, IRESs are commonly used to direct the expression of the second cistrons of bicistronic mRNAs. The mechanism of action of IRESs is not fully understood and a certain number of laboratories were not successful in using them in a reliable manner. Three observations done in our laboratory suggested that IRESs might not work as functionally as it was generally believed. Stem loops added before IRESs inhibited mRNA translation. When added into bicistronic mRNAs, IRESs initiated translation of the second cistrons efficiently only when the intercistronic region contained about 80 nucleotides, and they did not work any more effectively with intercistronic regions containing at least 300-400 nucleotides. Conversely, IRESs inserted at any position into the coding region of a cistron interrupted its translation and initiated translation of the following cistron. The first two data are hardly compatible with the idea that IRESs are able to recruit ribosomes without using the classical scanning mechanism. IRESs are highly structured and cannot be scanned by the 40S ribosomal subunit. We suggest that IRESs are short-circuited and are essentially potent stimulators favoring translation in particular physiological situations.

Beneficial effects of fibrates on apolipoprotein A-I metabolism occur independently of any peroxisome proliferative response.

BACKGROUND: In humans, fibrates are frequently used normolipidemic drugs. Fibrates act by regulating genes involved in lipoprotein metabolism via activation of the peroxisome proliferator-activated receptor-alpha (PPARalpha) in liver. In rodents, however, fibrates induce a peroxisome proliferation, leading to hepatomegaly and possibly hepatocarcinogenesis. Although this peroxisome proliferative response appears not to occur in humans, it remains controversial whether the beneficial effects of fibrates on lipoprotein metabolism can occur dissociated from such undesirable peroxisomal response. Here, we assessed the influence of fenofibrate on lipoprotein metabolism and peroxisome proliferation in the rabbit, an animal that, contrary to rodents and similar to humans, is less sensitive to peroxisome proliferators. METHODS AND RESULTS: First, we demonstrate that in normal rabbits, fenofibrate given at a high dose for 2 weeks does not influence serum concentrations or intestinal mRNA levels of the HDL apolipoprotein apoA-I. Therefore, the study was continued with human apoA-I transgenic rabbits that overexpress the human apoA-I gene under control of its homologous promoter, including its PPAR-response elements. In these animals, fenofibrate increases serum human apoA-I concentrations via an increased expression of the human apoA-I gene in liver. Interestingly, liver weight or mRNA levels and activity of fatty acyl-CoA oxidase, a rate-limiting and marker enzyme of peroxisomal beta-oxidation, remain unchanged after fenofibrate. CONCLUSIONS: Expression of the human apoA-I transgene in rabbit liver suffices to confer fibrate-mediated induction of serum apoA-I. Furthermore, these data provide in vivo evidence that the beneficial effects of fibrates on lipoprotein metabolism occur mechanistically dissociated from any deleterious activity on peroxisome proliferation and possibly hepatocarcinogenesis.

Human epidermal keratinocytes upregulate expression of the prolactin receptor after the onset of terminal differentiation, but do not respond to prolactin.

Growing and differentiating keratinocytes maintain the epidermal barrier. This is partly controlled by growth factors and hormones. Prolactin (PRL) is named after its hormonal role in mammals during lactation, but is found in all vertebrates where PRL exerts various effects. In serum-free keratinocyte cultures, PRL was thought to be the factor responsible for the proliferative effect of bovine pituitary extract. Here, we evaluated PRL as a clonogenic factor for keratinocytes and found no mitogenic activity. Studying the expression of the PRL receptor by keratinocytes, we found the receptor upregulated only after culture confluence, in differentiating keratinocytes, but we were unable to detect any cellular response to PRL. The hormone does not alter the gene expression of either early (suprabasal keratin) or late (involucrin) differentiation markers by keratinocytes. Accordingly, no activation of the transcription factor Stat5 by PRL can be detected in keratinocytes, Stat5 being nevertheless detected by Western blot.

Effect of intercistronic length on internal ribosome entry site (IRES) efficiency in bicistronic mRNA.

Specific structures found in the mRNA of picornavirus are known to allow a cap-independent translation. These structures, named internal ribosome entry sites (IRES), are also able to favor translation of the second cistron in bicistronic mRNAs. Their mechanism of action is not well understood. In the present study, two IRESs have been used: the IRES from poliovirus and a newly discovered IRES (SUR) composed of the 5' P untranslated sequence from SV40 early genes, the R structure, and a small part of the U5 region from the human leukemia virus-1 (HTLV-1). The bicistronic constructs containing the firefly luciferase gene as the first cistron and the chloramphenicol acetyltransferase (CAT) as the second cistron were driven by the Rous sarcoma virus (RSV) promoter and contained the early gene SV40 terminator. All the resulting plasmids were tested by transfection in HeLa and CHO cells. In the bicistronic mRNAs without IRES, the expression of the CAT gene was dependent on the distance between the two cistrons. The maximum efficiency in the expression of the second cistron was obtained when the intercalating RNA was composed of 30 to 90 nucleotides. This expression was deeply reduced when the intercalating fragment contained 8 or 300 nucleotides and was undetectable with 500 nucleotides. Unexpectedly, the luciferase mRNA was almost not expressed when the intercalating RNA was of 8 or 30 nucleotides. Expression of the luciferase gene occurred when the intercistronic RNA fragment was of 80 nucleotides and it became lower at 300 and 500 nucleotides. The same observations were done when the poliovirus or the SUR IRESs were added after the intercistronic spacers. However, expression of the CAT gene was amplified by both IRESs. When the CAT cistron preceded by the poliovirus or SUR IRES was introduced within luciferase cistron, 316 nucleotides before its termination codon, the IRESs were able to initiate translation of the following CAT gene irrespectively of the mRNA luciferase reading frame. Moreover, with all these constructs the highest expression level of the CAT cistron did not exceed 10% of that obtained with the same vector carrying only the CAT cistron. To identify a possible relation between the IRESs and the cap site, the CAT cistron preceded or not with an IRES was introduced 210 nucleotides downstream of the AUG codon of the luciferase gene (i.e., 258 nucleotides from the cap site) and 100 nucleotides after an added UAG termination codon. Expression of the CAT gene was not modified by the addition of the poliovirus IRES but it was strongly stimulated by the SUR IRES (the level of expression corresponded to 65% of that obtained with the same vector carrying only the CAT cistron). These results suggest that there is a cooperation between the cap and the SUR IRES and not the poliovirus IRES to stimulate translation. These data indicate that IRESs must be introduced in precise position to allow an efficient expression of the second cistron in bicistronic mRNAs.

Cloning, structural organization and tissue-specific expression of the rabbit transferrin gene.

We cloned the rabbit transferrin (rTf) cDNA and gene, and quantified the expression of the rTf gene at the RNA level in various organs. The tissue-specific pattern of expression of rTf gene is different to those in other species, with a high expression in mammary gland and kidney. The exon/intron structure of the rTf gene (17 exons/16 introns) is similar to those of transferrins from other species. The sequence of the rTf cDNA already published is corrected and lengthened in the 5' region, and a likely polymorphism is documented.