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PubMed1994

Comparative responses of genetically lean and fat chickens to lysine, arginine and non-essential amino acid supply. I. Growth and body composition.

1. Three experiments were performed to study the effects of amino acid imbalance on the growth of genetically lean (LL) or fat (FL) male chickens from 28 to 42 d of age. In experiment 1, five concentrations of digestible lysine were compared (4.75, 6.75, 7.75, 8.75 and 9.75 g/kg). In experiment 2, four concentrations of digestible arginine were compared (6.53, 7.69, 8.84 and 10.0 g/kg). In experiment 3, three diets were compared: a high-protein diet (189 g CP/kg), a low-protein diet containing added essential amino acids (EAA) (144 g CP/kg) and this low-protein diet supplemented with 40 g/kg of non-essential amino acids (NEAA) (glutamic acid+aspartic acid). 2. LL birds exhibited a lower growth rate than the FL when the diet was deficient in either lysine or arginine. Plotting weight gain against lysine or arginine intake suggested that most of this effect was caused by variations in food intake. 3. When protein gains (body or total proteins) were plotted against lysine or arginine intake, LL chickens appeared more efficient than FL chickens. 4. Similar growth rates, although slightly lower in FL, were obtained with low- and high-protein diets. However, NEAA supplementation of the low-protein diet reduced adiposity of LL and did not modify that of FL. Increasing crude protein content (all amino acids) was more effective than NEAA supplementation in decreasing the adiposity of both lines.

Amino Acids↗

PubMed2002

Pharmacological characterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats.

Chronic intracerebroventricular (ICV) treatment with nociceptin/orphanin FQ (NC), the endogenous ligand for the opioid receptor-like 1 (ORL1) receptor, reduces ethanol intake in alcohol-preferring rats and abolishes the rewarding properties of ethanol in the place conditioning paradigm. To pharmacologically characterize the receptor involved, the present study evaluated the effect on ethanol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats of ICV injections for 8 days of NC or of the NC analogs NC(1-17)NH(2), NC(1-13)NH(2), NC(1-12)NH(2) and [Nphe(1)]NC(1-13)NH(2). In vitro studies indicate that NC, NC(1-17)NH(2), NC(1-13)NH(2) and NC(1-12)NH(2) are agonists, while [Nphe(1)]NC(1-13)NH(2) is a selective antagonist at the ORL1 receptor. Freely feeding and drinking rats were offered 10% ethanol 30 min/day at the beginning of the dark phase of the light cycle. NC significantly attenuated ethanol intake at 500 or 1000 ng/rat (210 or 420 pmol/rat). NC(1-17)NH(2), markedly reduced ethanol intake, but its effect was statistically significant at 1000 (420 pmol/rat), not at 500 ng/rat (210 pmol/rat). After the end of treatment ethanol drinking promptly came back to baseline level. Ethanol consumption was also reduced by NC(1-13)NH(2); however, its effect was less potent and pronounced. NC(1-12)NH(2) did not modify ethanol intake at doses up to 4000 ng/rat (2339 pmol/rat). Water and food consumption were not modified. Treatment with [Nphe(1)]NC(1-13)NH(2), 66 or 99 microg/rat, did not modify ethanol intake; however, [Nphe(1)]NC(1-13)NH(2), 66 microg/rat, given just before 1000 ng/rat of NC(1-17)NH(2), abolished the effect of the agonist. The present results show that the 13 amino acid N-terminal sequence of NC is essential for the effect on ethanol intake and indicate that [Nphe(1)]NC(1-13)NH(2) acts as an antagonist to block the effect of NC. These findings provide further evidence that selective agonists at the ORL-1 receptor attenuate ethanol intake in alcohol-preferring rats and suggest that the NC/ORL1 system may represent an interesting target for treatment of alcohol abuse.

Alcohol Drinking↗

PubMed1997

Transformation of wheat with high molecular weight subunit genes results in improved functional properties.

The high molecular weight (HMW) subunits of wheat glutenin are major determinants of the elastic properties of gluten that allow the use of wheat doughs to make bread, pasta, and a range of other foods. There are both quantitative and qualitative effects of HMW subunits on the quality of the grain, the former being related to differences in the number of expressed HMW subunit genes. We have transformed bread wheat in order to increase the proportions of the HMW subunits and improve the functional properties of the flour. A range of transgene expression levels was obtained with some of the novel subunits present at considerably higher levels than the endogenous subunits. Analysis of T2 seeds expressing transgenes for one or two additional HMW subunits showed stepwise increases in dough elasticity, demonstrating the improvement of the functional properties of wheat by genetic engineering.

Glutens↗

PubMed2006

A leaf-based regeneration and transformation system for maize (Zea mays L.).

Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic and applied research. While being the world's major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants.

DNA, Plant↗

PubMed1999

Safety assessment of transgenic potatoes with soybean glycinin by feeding studies in rats.

Feeding studies of transgenic potatoes with native and designed soybean glycinins in rats were done for four weeks. The designed glycinin has four additional methioninyl residues in the middle of the glycinin molecule. Rats were divided into four groups fed (I) only a commercial diet, (II) the diet plus non-transgenic potatoes, (III) the diet plus transgenic potatoes with native glycinin, and (IV) the diet plus transgenic potatoes with designed glycinin. Rats were fed 2,000 mg/kg-weight potatoes every day by oral administration. During the period tested, rats in each group (groups II, III, and IV) grew well without marked differences in appearance, food intake, body weight, or in cumulative body weight gain. No significant differences were also found in blood count, blood composition, and in internal organ weights among the rats after feeding potatoes (groups II, III, and IV) for four weeks. Necropsy at the end of experiment indicated neither pathologic symptoms in all rats tested nor histopathological abnormalities in liver and kidney. Judging from these results, the transgenic potatoes with glycinins are confirmed to have nearly the same nutritional and biochemical characteristics as non-transgenic one.

Animal Feed↗

PubMed2004

Identification of major quantitative trait loci controlling body weight variation in ob/ob mice.

The adipocyte hormone leptin constitutes an important component of the regulation of energy homeostasis; leptin-deficient animals, such as obese mice, are strikingly overweight. The seemingly uninhibited weight gain in obese mice belies the fact that control of energy homeostasis remains under precise, heritably modifiable control. Herein, we report large, heritable differences in body weight and food intake between BTBR-ob/ob and B6-ob/ob mice. We have identified two loci, called modifier of obese (Moo1 and Moo2), that explain the majority of the heritable variance in (BTBR x B6) F(2)-ob/ob mice. Using interval-specific congenic mouse lines, we mapped Moo1 to an 8-Mb segment of chromosome 2 and demonstrated that Moo1 exerts its effects primarily by regulating total fat mass. Although null alleles of leptin are rare, the majority of overweight adults are leptin resistant, suggesting that leptin-independent pathways, such as those studied here, are important regulators of energy homeostasis. Thus, the identification of these loci may provide important new insights into the pathogenesis of human obesity.

Aging↗

PubMed2003

[Transgenic bioinsecticides inimical to parasites, but imical to environment].

Identification of Bacillus thuringiensis (Bt) parasporal crystalline inclusions composed of Cry proteins (=delta-endotoxins) resulted in introduction of microbial pesticides for biological control of some parasites. Delta-endotoxins are encoded by cry genes and are active against pest and nuisance insects (mostly mosquitoes and black flies--vectors of still important infectious diseases). The recent significant progress in DNA recombination technique may overcome limitations (a short residual persistence and a narrow spectrum of activity) associated with application of Bt conventional products. An introduction of cry genes from mosquitocidal subspecies B. th. israelensis (Bti) to the aquatic microorganisms inhabiting the same water bodies as mosquito and fly larvae (Diptera), has considerably improved the toxin delivery system to target insects. However, in the first experiments, in which Bti genes were cloned in cyanobacteria (Agmenellum quadruplicatum, Synechocystis PCC6803), a low gene expression was observed. Thus, it was necessary to integrate cry genes with strong promoters or to increase the number of vector-introduced copies. To overcome the obstacles of low gene expression and regulatory restriction for recombinant organisms, Bti spore/crystal formulations were encapsulated in the aquatic protozoan, Tetrahymena pyriformis. Large numbers of crystals (180 to 240/cell) were accumulated in its food vacuoles. This system resulted also in an increase in toxin persistence from 24 to 71 h. Cloning Bti genes in B. sphaericus (which also produces mosquitocidal proteins) was another way of an increasing Bt crystal residual activity. In this case, the crystals were additionally protected by B. sphaericus exosporium. These transgenic bacteria produced large amounts of delta-endotoxins that remained under water surface longer than the wild B. sphaericus strains. Moreover, they had a broader spectrum of insecticidal activity, because B. sphaericus is toxic mostly to Culex and Anopheles, and Bti--mostly to Culex, Aedes and some Simmulidae. Gram-negative bacteria (Asticcacaulis excentricus, Caulobacter crescentus and Ancylobacter aquaticus) turned out also to be effective delta-endotoxin producers. They grow on simple media and do not contain proteases which could degrade Cry proteins. In some cases, 100% mosquito larvae mortality was observed as a result of an exposure to transgenic microorganisms containing Bti genes. However, transgenic techniques are still not very popular in the world, despite their efficacy in biological control of insects. The transgenic organism construction is expensive and time-consuming. Genetic engineering is still raising a lot of anxieties and doubts concerning inappropriate use of modified organisms. On the other hand, this technology could solve many problems associated with vectors of important diseases, which are still unapproachable to contemporary medicine.

Animals↗

PubMed1997

Diet, nutrition and prostate cancer.

Although much has been written, little is known about the causes of prostate cancer. Variations between populations in the incidence of invasive cancers, together with changes in the incidence of invasive cancers in migrants, have pointed to environmental (lifestyle) factors that might be amenable to intervention. Conversely, there is a lack of international variation in the prevalence of microscopic tumours, so the essential question is: what causes only some of the common microscopic tumours to become aggressive? Dietary factors hold the most promise in this regard and have been the subject of recent reviews. The strongest and most consistent effects are positive associations with animal products such as red meats, eggs and dairy foods, and possibly by implication, fat. Evidence of a protective effect of fruit and vegetables is weak and inconsistent, as is the relationship with vitamin A and carotenoids, such as beta-carotene. There are some interesting leads. Lycopene, the carotenoid found in tomatoes, has been reported to be protective; alpha-tocopherol supplementation has shown a protective effect in one intervention study; and vitamin D has been shown to be protective in a prospective study. Interest is also growing in phytoestrogens and the extent to which dietary manipulation with these and other phytochemicals might influence prostate cancer by modifying male sex hormone levels or actions. There is limited evidence of associations with obesity. It is not known whether these are related to a particular dietary pattern or to possible physiological effects on the male's hormonal milieu. Associations with lean body mass are likely to be related to the action of androgens during growth and development. Dietary and nutritional effects on prostate cancer do not appear to be strong, but they may be subtle and attenuated by measurement error. To explore these aspects further will require large prospective studies that include improved (repeated) dietary measurements and also blood sampling, so that genetic polymorphisms can be adequately investigated. Such studies are underway.

Alcohol Drinking↗

PubMed2006

Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification.

To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.

Actins↗

PubMed2006

Re-introducing bacteria in mosquitoes--a method for determination of mosquito feeding preferences based on coloured sugar solutions.

In this study, sugar-feeding was investigated as a possible means of re-introducing bacteria into mosquito midguts with the aim of identifying bacteria that are suitable for creating paratransgenic mosquitoes. In a paratransgenic approach, bacteria are utilised to deliver effector molecules capable of inhibiting pathogen development in the midgut of the vector. To determine if mosquitoes discriminate between sterile sugar solutions and sugar solutions with bacteria, a method for screening mosquito feeding preferences was developed. This method was tested for Aedes aegypti, Anopheles arabiensis and An. gambiae s.s. mosquitoes and is based on a dual-choice test of solutions labelled with food dyes. Three different tests (dye/colour detection, sugar detection and sugar-concentration detection) were performed to evaluate the method, after which bacteria previously isolated from mosquitoes were used in the experiments. It was shown that mosquitoes do not discriminate between sugar solutions with or without these bacteria indicating that sugar-feeding is a possible means to introduce bacteria into mosquitoes. Furthermore, two different setups of the method were used, enabling us to differentiate between tactile/taste and olfactory responses. The method described in this paper is easy to use, cost-effective and allows broad screening of mosquito sugar-feeding preferences.

Aedes↗

PubMed2002

Molecular pathways to obesity.

Obesity results from a chronic imbalance between energy intake and energy expenditure. Environmental factors, such as the increased availability of high caloric food or the decreased need for physical activity, contribute to its development and their influence is amplified by genetic predisposition. In recent years remarkable progress has been made in the understanding of the pathophysiology of obesity. Although most of the insights into the regulation of energy balance have been obtained in rodent models, the rare clinical cases of monogenic obesity provided evidence for the importance of several of these mechanisms in humans. The identification of leptin as a factor originating from adipose tissue and informing the brain about the status of energy reserves firmly established the concept of long-term regulation of body fat stores. The disappointing therapeutic results with leptin in obese patients could be explained by the fact that during evolution this hormone developed rather as a starvation signal than as an adiposity signal. It is conceivable that the pharmacological interference with mechanisms downstream of leptin, for example with the melanocortin pathway, might be therapeutically more promising. The discovery of new molecular mechanisms involved in the regulation of the differentiation and proliferation of adipocytes and the elucidation of their paracrine and endocrine functions have changed the traditional view of adipose tissue as an inert depot for triglycerides. The identification of new uncoupling proteins could modify the current concepts of the regulation of thermogenesis in humans. The remarkable progress in the identification of novel targets involved in the regualtion of energy balance should have a positive impact on the search for new antiobesity agents.

Animals↗

PubMed2002

Life span extension by reduction in growth hormone-insulin-like growth factor-1 axis in a transgenic rat model.

The longer life span in dwarf mice suggests that a reduction in the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis retards aging and extends the life span in mammals. We tested this hypothesis in a transgenic strain of rats whose GH gene was suppressed by an anti-sense GH transgene. Male rats homozygous for the transgene (tg/tg) had a reduced number of pituitary GH cells, a lower plasma concentration of IGF-1, and a dwarf phenotype. Heterozygous rats (tg/-) had an intermediate phenotype in plasma IGF-1, food intake, and body weight between tg/tg and control (-/-) rats. The life span of tg/tg rats was 5 to 10% shorter than -/- rats. In contrast, the life span of tg/- rats was 7 to 10% longer than -/- rats. Pathological analysis suggested that neoplasms caused earlier death in tg/tg rats; in contrast, tg/- rats had reduced nonneoplastic diseases and a prolonged life span. Immunological analysis revealed a smaller population and lower activity of splenic natural killer cells in tg/tg rats. The results of the present study support the hypothesis, but suggest that there is an optimal level of the GH-IGF-1 axis to maximize survival in mammals.

Animals↗

PubMed2002

Increasing antioxidant levels in tomatoes through modification of the flavonoid biosynthetic pathway.

Flavonoids are a diverse group of phenolic secondary metabolites that occur naturally in plants and therefore form an integral component of the human diet. Many of the compounds belonging to this group are potent antioxidants in vitro and epidemiological studies suggest a direct correlation between high flavonoid intake and decreased risk of cardiovascular disease, cancer and other age-related diseases. Enhancing flavonoid biosynthesis in chosen crops may provide new raw materials that have the potential to be used in foods designed for specific benefits to human health. Using genetic modification, it was possible to generate several tomato lines with significantly altered flavonoid content and to probe the role and importance of several key enzymatic steps in the tomato flavonoid biosynthetic pathway. Most notably an up to 78-fold increase in total fruit flavonols was achieved through ectopic expression of a single biosynthetic enzyme, chalcone isomerase. In addition, chalcone synthase and flavonol synthase transgenes were found to act synergistically to up-regulate flavonol biosynthesis significantly in tomato flesh tissues.

Acyl Coenzyme A↗

PubMed2002

Metabolic engineering of amino acids and storage proteins in plants.

Amino acid pathways are important targets for plant metabolic engineering. Since plants represent the major global food supply, large efforts are devoted to increasing the content of "essential" amino acids, which are absolutely required in human foods and animal feeds. Engineering of amino acids is also undertaken to improve plant growth and stress tolerance. Many of the pathways of amino acid metabolism in plants have been elucidated, and genes encoding most of the enzymes are now available. The expression of recombinant genes in transgenic plants, coupled with genetic and biochemical approaches, has contributed significantly to the understanding of regulatory networks of the metabolism of amino acids and their incorporation into proteins. This knowledge is now being extensively applied to metabolic engineering of crops, and this is reflected by a large patent literature. The problems of engineering plant amino acid metabolism, and ways to solve them, are discussed using the essential amino acids lysine and methionine as examples.

Amino Acids, Essential↗

PubMed2005

Gluten: a two-edged sword. Immunopathogenesis of celiac disease.

Celiac disease (CD) is a small intestinal disorder caused by adaptive and innate immune responses triggered by the gluten proteins present in wheat. In the intestine, gluten is partially degraded and modified, which results in gluten peptides that bind with high affinity to HLA-DQ2 or HLA-DQ8 and trigger an inflammatory T cell response. Simultaneously, gluten exposure leads to increased production of IL15, which induces the expression of NKG2D on intraepithelial lymphocytes and its ligand MICA on epithelial cells, leading to epithelial cell destruction. The gluten-specific T cell response results in the production of antibodies against tissue transglutaminase and these are specific indicators of disease. CD is one of the most common inherited diseases, the HLA-DQ locus being the major contributing genetic factor. However, as the inheritance does not follow a Mendelian segregation pattern, multiple other genes, each with relative weak effect, contribute to disease development. An important role for environmental factors, however, can not be ignored as the concordance rate in monozygous twins is considerably less than 100%. The identification of these environmental factors and susceptibility genes may allow a better understanding of disease etiology and provide diagnostic and prognostic markers. The current treatment for CD consists of a life-long gluten-free diet. Although long thought to be impossible, recent results suggest that the development of nontoxic wheat varieties may be feasible, which would aid disease prevention and provide an alternative food source for patients.

Autoantibodies↗

PubMed2005

Food safety evaluation of crops produced through genetic engineering--how to reduce unintended effects?

Scientists started applying genetic engineering techniques to improve crops two decades ago; about 70 varieties obtained via genetic engineering have been approved to date. Although genetic engineering offers the most precise and controllable genetic modification of crops in entire history of plant improvement, the site of insertion of a desirable gene cannot be predicted during the application of this technology. As a consequence, unintended effects might occur due to activation or silencing of genes, giving rise to allergic reactions or toxicity. Therefore, extensive chemical, biochemical and nutritional analyses are performed on each new genetically engineered variety. Since the unintended effects may be predictable on the basis of what is known about the insertion place of the transgenic DNA, an important aim of plant biotechnology is to define techniques for the insertion of transgene into the predetermined chromosomal position (gene targeting). Although gene targeting cannot be applied routinely in crop plants, given the recent advances, that goal may be reached in the near future.

Consumer Product Safety↗

PubMed2004

Vaccination with genetically engineered allergens prevents progression of allergic disease.

IgE-mediated allergy affects >25% of the population in industrialized countries. Repeated contact with the disease-eliciting allergens induces rises of allergen-specific IgE Abs and progression of the disease to more severe manifestations. Our study uses a type of vaccine that is based on genetically modified allergen derivatives to treat allergic patients. We developed hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, by genetic engineering and vaccinated birch pollen-allergic patients (n = 124) in a double-blind, placebo-controlled study. Active treatment induced protective IgG Abs that inhibited allergen-induced release of inflammatory mediators. We also observed a reduction of cutaneous sensitivity as well as an improvement of symptoms in actively treated patients. Most important, rises of allergen-specific IgE induced by seasonal birch pollen exposure were significantly reduced in vaccinated patients. Vaccination with genetically engineered allergen derivatives is a therapy for allergy that not only ameliorates allergic reactions but also reduces the IgE production underlying the disease.

Allergens↗

PubMed2003

On the safety of a new generation of DSM Aspergillus niger enzyme production strains.

Consumers safety of enzyme preparations is determined by three variables: the producing organism, the raw materials used in the production, and the production process itself. The latter one is embedded in current Good Manufacturing Practice (cGMP) and Hazard Analysis of Critical Control Points (HACCP); therefore the safety focus can be directed to raw materials and the producing organism. In this paper, we describe the use of novel genetically modified strains of Aspergillus niger-made by a design and build strategy-from a lineage of classically improved strains with a history of safe use in enzyme production. The specifics of the host strain allow for integration and over-expression of any gene of interest at a targeted integration site implying that the rest of the host genome is not affected by this integration. Furthermore due to the fact that the newly integrated gene copies are put under the genetic regulation of the host's own glucoamylase promoter, the recipe of the production process of any new production strain can be kept constant with respect to the raw materials composition. Consequently the safety of a new enzyme product from these novel genetically modified strains is determined by the background of the production organism. The use of a strain with a history of safe use and targeted integration according to the concept described above has consequences for the safety studies on the final product. If a known enzymatic activity is over-expressed the safety of a new enzyme preparation is covered by the results of the safety studies performed for other strains from this specific Aspergillus niger strain lineage. In this paper an overview is given on the available toxicity tests with these strains. We conclude that for new enzyme products produced with strains from this lineage using the design and build technology no new sub-acute/chronic oral toxicity studies are needed. This also has the benefit that no longer test animals are needed to demonstrate the safety of products produced by these strains.

Animals↗