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PubMed1995

Yeasts: from genetics to biotechnology.

Yeasts have been known and used in food and alcoholic fermentations ever since the Neolithic Age. In more recent times, on the basis of their peculiar features and history, yeasts have become very important experimental models in both microbiological and genetic research, as well as the main characters in many fermentative production processes. In the last 40 years, advances in molecular biology and genetic engineering have made possible not only the genetic selection of organisms, but also the genetic modification of some of them, especially the simplest of them, such as bacteria and yeasts. These discoveries have led to the availability of new yeast strains fit to fulfill requests of industrial production and fermentation. Moreover, genetically modified and transformed yeasts have been constructed that are able to produce large amounts of biologically active proteins and enzymes. Thus, recombinant yeasts make it easier to produce drugs, biologically active products, diagnostics, and vaccines, by inexpensive and relatively simple techniques. Yeasts are going to become more and more important in the "biotechnological revolution" by virtue of both their features and their very long and safe use in human nutrition and industry.

Biotechnology↗

PubMed1998

Drinking patterns in genetic low-alcohol-drinking (LAD) rats after systemic cyanamide and cerebral injections of THP or 6-OHDA.

A key question related to the role of acetaldehyde and aldehyde adducts in alcoholism concerns their relationship to the genetic mechanisms underlying drinking. Experimentally, the low-alcohol-drinking (LAD) rat represents a standard rodent model having a strong aversion to alcohol. In these experiments, preferences for water vs. alcohol, offered in concentrations from 3% to 30%, were determined over 10 days in adult LAD rats (N = 6 per group). Then a saline vehicle or either 10 or 20 mg/kg of the aldehyde dehydrogenase (AIDH) inhibitor, cyanamide, was injected s.c. twice daily for 3 days. Secondly, either 0.5 or 1.0 microg of tetrahydropapaveroline (THP) was infused i.c.v. twice daily for 3 days in LAD rats (N = 8) and, as a genetic control, THP also was infused identically in Sprague-Dawley (SD) rats (N = 8). The results showed that the lower and higher doses of cyanamide augmented alcohol intakes in 33% and 50% of the LAD rats, respectively, with the patterns of drinking resembling that of genetic high-alcohol-drinking HAD or P rats. Although i.c.v. infusions of THP had little effect on alcohol preference of LAD rats, alcohol drinking was enhanced significantly in the SD rats. In a supplementary study, 200 microg of 6-hydroxydopamine (6-OHDA) also was infused i.c.v. in LAD rats (N = 7) on two consecutive days; no change occurred in the characteristic aversion to alcohol. These findings suggest that in certain individuals, a perturbation in the synthesis of AIDH can modify the genetically based aversion to alcohol, thus precipitating the liability for alcoholism. In that neither THP nor 6-OHDA lesioning exerted any effect on the genetic nondrinking LAD animal suggests that an unknown endogenous factor in the brain must underlie the cyanamide-induced shift to alcohol preference. We conclude that the genetic elements that normally prevent the progression to addictive drinking in most individuals appear to be invariant and irreversible.

Alcohol Drinking↗

PubMed2005

Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants.

Transgenic plant varieties, which are tolerant to glufosinate-ammonium, were developed. The herbicide tolerance is based upon the presence of either the bar or the pat gene, which encode for two homologous phosphinothricin acetyltransferases (PAT), in the plant genome. Based on both a review of published literature and experimental studies, the safety assessment reviews the first step of a two-step-approach for the evaluation of the safety of the proteins expressed in plants. It can be used to support the safety of food or feed products derived from any crop that contains and expresses these PAT proteins. The safety evaluation supports the conclusion that the genes and the donor microorganisms (Streptomyces) are innocuous. The PAT enzymes are highly specific and do not possess the characteristics associated with food toxins or allergens, i.e., they have no sequence homology with any known allergens or toxins, they have no N-glycosylation sites, they are rapidly degraded in gastric and intestinal fluids, and they are devoid of adverse effects in mice after intravenous administration at a high dose level. In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the PAT proteins in human food or in animal feed.

Acetyltransferases↗

PubMed2003

Identification of transglutaminase-mediated deamidation sites in a recombinant alpha-gliadin by advanced mass-spectrometric methodologies.

Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was to achieve a detailed structural characterization of modifications induced by transglutaminase on gliadin peptides. Therefore, structural analyses were carried out on a recombinant alpha-gliadin and on a panel of 26 synthetic peptides, overlapping the complete protein sequence. Modified glutamine residues were identified by means of advanced mass-spectrometric methodologies on the basis of MALDI-TOF-MS and tandem mass spectrometry. Results led to the identification of 19 of 94 glutamine residues present in the recombinant alpha-gliadin, which were converted into glutamic acid residues by a transglutaminase-mediated reaction. This allowed us to achieve a global view of the modifications induced by the enzyme on this protein. Furthermore, results gathered could likely be utilized as relevant information for a better understanding of processes leading to T-cell recognition of gliadin peptides involved in celiac disease.

Amides↗

PubMed1992

Stress resistance of Drosophila transgenic for bovine CuZn superoxide dismutase.

Several oxidative and non-oxidative stresses were applied to two transgenic strains of Drosophila melanogaster (designated P(bSOD)5 and P(bSOD)11) that express superoxide dismutase (SOD) at elevated levels, and control strains that express normal SOD levels. Transgenic strain P(bSOD)5 exposed to paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride), a redox cycling agent that generates superoxide anion when metabolized in vivo, was significantly more resistant to this xenobiotic than control flies. When test flies were subjected to 100% oxygen for 20 min each day, the mean lifespan was 3.62 days for control strain 25, but 4.35 days for both transgenic strains. The mortality curves of strains fed 1% H2O2 were similar, but the median lifespan of 72 h for controls and 64 h for transgenics suggests that the transgenic flies were slightly more sensitive to H2O2. The activity of catalase was the same for all strains. Using starvation resistance as a non-oxidative stress, flies maintained on water without any food had identical survival curves; for all strains, the median lifespan was 72 h. Throughout the lifespan, no statistically significant difference in physical activity was displayed for transgenic versus control flies. Collectively, these data suggest that the increased lifespan previously observed in SOD transgenics is specifically related to resistance to oxidative stresses.

Animals↗

PubMed2002

Position of the American Dietetic Association: weight management.

It is the position of the American Dietetic Association that successful weight management to improve overall health for adults requires a lifelong commitment to healthful lifestyle behaviors emphasizing sustainable and enjoyable eating practices and daily physical activity. Americans are increasing in body fat as they become more sedentary. Obesity has reached epidemic proportions and health care costs associated with weight-related illnesses have escalated. Although our knowledge base has greatly expanded regarding the complex causation of increased body fat, little progress has been made in long-term maintenance interventions with the exception of surgery. Lifestyle modifications in food intake and exercise remain the hallmarks of effective treatment, but are difficult to initiate and sustain over the long term. The dietitian can play a pivotal role in modifying weight status by helping to formulate reasonable goals which can be met and sustained with a healthy eating approach as outlined in the Dietary Guidelines for 2000. Any changes in dietary intake and exercise patterns which decrease caloric intake below energy expenditure will result in weight loss, but it is the responsibility of the dietitian to make sure the changes recommended are directed toward improved physiological and psychological health. A thorough clinical assessment should help define possible genetic, environmental, and behavioral factors contributing to weight status and is important to the formulation of an individualized intervention. The activation of treatment strategies is often limited by available resources and cost. Reimbursement by third party payers for services is limited. Health care dollars are consumed for treatment of weight-related diseases. Public policy must change if the obesity epidemic is to be stopped and appropriate weight management techniques activated.

Body Weight↗

PubMed2007

Early life exposure to antibiotics and the subsequent development of eczema, wheeze, and allergic sensitization in the first 2 years of life: the KOALA Birth Cohort Study.

OBJECTIVES: Antibiotic exposure in early life may be associated with atopic disease development either by interfering with bacterial commensal flora or by modifying the course of bacterial infections. We evaluated early life exposure to antibiotics and the subsequent development of eczema, wheeze, and allergic sensitization in infancy. METHODS: Information on antibiotic use in the first 6 months and eczema and wheeze until age 2 was collected by repeated questionnaires in 2764 families participating in the KOALA (Child, Parent and Health: Lifestyle and Genetic Constitution [in Dutch]) Birth Cohort Study in The Netherlands. Antibiotic intake was evaluated both as maternal antibiotic use during breastfeeding and infant oral medication. Venous blood samples taken from 815 infants at 2 years of age were analyzed for total and specific immunoglobulin E against common food and inhalant allergens using a radioallergosorbent test. Multivariate logistic regression analysis was used to adjust for confounding factors. RESULTS: During the first 2 years, eczema was present in 32% of all infants, recurrent wheeze in 11%, and prolonged wheezing in 5%. At 2 years old, 27% of children were sensitized against > or = 1 allergen. At 6 months old, 11% had been exposed to antibiotics through breast milk and 20% directly through medication. The risk for recurrent wheeze, and prolonged wheeze was higher in infants directly exposed to antibiotics through medication, also after excluding from the analyses children who wheezed in the same period as an antibiotic had been used (avoiding reverse causation). Antibiotic use through breastfeeding was associated with recurrent wheeze, but prolonged wheeze was not. Eczema and sensitization were not associated with antibiotic exposure. CONCLUSIONS: We demonstrated that early antibiotic use preceded the manifestation of wheeze but not eczema or allergic sensitization during the first 2 years of life. Different biological mechanisms may underlie the etiology of wheeze compared with eczema or sensitization. Antibiotic exposure through breastfeeding enhanced the risk for recurrent wheeze, but this needs further confirmation.

Anti-Bacterial Agents↗

PubMed2002

A long way ahead: understanding and engineering plant metal accumulation.

Some plants can hyperaccumulate metal ions that are toxic to virtually all other organisms at low dosages. This trait could be used to clean up metal-contaminated soils. Moreover, the accumulation of heavy metals by plants determines both the micronutrient content and the toxic metal content of our food. Complex interactions of transport and chelating activities control the rates of metal uptake and storage. In recent years, several key steps have been identified at the molecular level, enabling us to initiate transgenic approaches to engineer the transition metal content of plants.

Biodegradation, Environmental↗

PubMed1995

Subcutaneous injections of the tachykinin senktide reduce alcohol intake in alcohol-preferring rats.

The present study evaluated the effect of SC injections of the selective NK3 tachykinin agonist, Suc-[Asp6,MePhe8]substance P(6-11), also referred to as senktide (SENK), on 8% alcohol intake in genetically selected alcohol-preferring rats. Animals were offered access to 8% ethanol for 2 h/day (between 1800 and 2000 h) and to tap water for 4 h/day (between 1800 and 2200 h); SENK was injected 10 min before access to fluids. The peptide significantly reduced alcohol intake at doses of 125 and 250 micrograms/kg, but not at 62.5 micrograms/kg. The reduction in alcohol intake was accompanied by a sharp increase in water intake, so that total fluid intake was never significantly modified. The same SC doses of SENK did not modify water intake in rats with access to water, as the only fluid, for 4 h/day. In food-deprived rats food intake was not altered by 125 micrograms/kg, whereas 250 micrograms/kg produced a reduction in food intake that was smaller in intensity and shorter lasting than the reduction in alcohol intake. The same doses of SENK did not modify 0.1% saccharin intake, nor did they elicit major competing behaviors. The results of the present study are in keeping with those obtained following central injection of NK3 agonists, and show that a behaviorally selective reduction of alcohol intake can be evoked also by peripheral administration of SENK.

Alcohol Drinking↗

PubMed2003

Genetically engineered probiotics.

Probiotic micro-organisms have been used for many years. Originating as food supplements, they are now most often administered orally and offer an attractive alternative for treating of intestinal disorders. A better understanding of the mechanisms by which these micro-organisms act has now opened up possibilities for designing new probiotic strains. Through genetic engineering, it is possible not only to strengthen the effects of existing strains, but also to create completely new probiotics. These need not necessarily be composed only of bacterial products but can also include elements of regulatory systems or enzymes derived from a foreign-human-source. If designed carefully and with absolute attention to biological safety in its broadest sense, the development of genetically modified probiotics has the potential to revolutionize alimentary health.

Animals↗

PubMed2002

Lantibiotics produced by lactic acid bacteria: structure, function and applications.

Lantibiotics are a diverse group of heavily modified antimicrobial and/or signalling peptides produced by a wide range of bacteria, including a variety of lactic acid bacteria. Based on their diverse structures and mode of action, at least six separate lantibiotic subgroups can be suggested, but all subgroups are characterized by significant post-translational modifications, which include the formation of (beta-methyl)lanthionines, among other unusual alterations. These small peptides are produced, modified, exported, sensed and combated by a complex set of proteins encoded by (usually) co-ordinately regulated operons. In some instances, the production and immunity have been shown to be auto-regulated by the mature lantibiotic. Since their discovery, interest in lantibiotics has been fuelled by their obvious potential as food-grade antimicrobials to improve food safety and quality; a potential which, to date, has been realised only by the longest characterised molecule, nisin. In addition, these peptides are often mooted as alternatives to antibiotics for some biomedical applications. The purpose of this paper is to review recent developments in our understanding of lantibiotic structure, molecular genetics and applications for this unusual class of bacteriocins.

Amino Acid Sequence↗

PubMed2001

Design and production of genetically modified soybean protein with anti-hypertensive activity by incorporating potent analogue of ovokinin(2-7).

The potent anti-hypertensive peptide, RPLKPW, has been designed based on the structure of ovokinin(2-7). The sequence encoding this peptide was introduced into three homologous sites in the gene for soybean beta-conglycinin alpha' subunit. The native alpha' subunit as well as the modified, RPLKPW-containing alpha' subunit were expressed in Escherichia coli, recovered from the soluble fraction and then purified by ion-exchange chromatography. The RPLKPW peptide was released from recombinant RPLKPW-containing alpha' subunit after in vitro digestion by trypsin and chymotrypsin. Moreover, the undigested RPLKPW-containing alpha' subunit given orally at a dose of 10 mg/kg exerted an anti-hypertensive effect in spontaneously hypertensive rats, unlike the native alpha' subunit. These results provide evidence for the first time that a physiologically active peptide introduced into a food protein by site-directed mutagenesis could practically function in vivo even at a low dose.

Administration, Oral↗

PubMed1995

Genetic engineering in plants.

Until now most research, and its funding, has been focused on animal and human health care as well as simple microbiological model systems such as Escherichia coli and yeast. Molecular plant studies have generally lagged behind, often simply adapting discoveries from the animal field to plants. Clearly, good health and the efficient tackling of diseases is crucial for the well-being of humans, and good remedies have a high economic value for the pharmaceutical industry. However, one should not forget that plants are an essential component of the large ecosystem that is our planet. They are not only the basic food producers but they are also necessary for a balanced atmosphere (oxygen production) and stable and viable climates. Especially in this period of demographic explosion and growing environmental deterioration, there is a need to rebuild our agricultural systems. Plants also have a wide variety of 'non-food' uses, for instance as energy sources, construction materials, or cosmetics. Last, but not least, they produce a lot of chemicals that can be used as pharmaceuticals. The growing awareness of the importance of plants has coincided with the development of plant molecular biology. Specific features make them ideally suited for gene engineering and genetic studies in general.

Biotechnology↗

PubMed1998

Molecular evidence for the gnathobasic derivation of arthropod mandibles and for the appendicular origin of the labrum and other structures.

Mandibles are feeding appendages functioning as "jaws" in the arthropod groups in which they occur. Which part of this appendage is involved in food manipulation (limb tip versus limb base), has been used to suggest phylogenetic relationships among some of the major taxa of arthropods (myriapods, crustaceans, and insects). As a way to independently verify the conclusions drawn from previous morphological analyses, we have studied the expression pattern of the gene Distal-less (Dll), which specifies the distal part of appendages. Our results show, in contrast to the traditional view, that both insect and crustacean adult mandibles are gnathobasic, handling food with the basal portion of the appendage. Furthermore, as is evident by the reduction in the number of Dll-expressing cells in the later developmental stages, adult diplopod jaws are also gnathobasic. Thus, jaws of all mandibulates (myriapods, crustaceans, and insects) seem to have a similar gnathobasic structure. We have also found that Dll is expressed in the labra of all arthropod taxa examined, suggesting that this structure is of appendicular derivation. Additionally, the spinnerets and book lungs of spiders, long considered on other grounds to be modified appendages, express Dll, confirming this interpretation. This study shows that, in addition to their use in phylogenetic and population genetic studies, molecular markers can be very useful for inferring the origins of a particular morphological feature.

Animals↗

PubMed1993

Metformin and brown adipose tissue thermogenetic activity in genetically obese Zucker rats.

The effect of chronic metformin treatment on brown adipose tissue thermogenetic activity was investigated in young genetically obese Zucker rats. The binding of [3H]GDP to brown adipose tissue mitochondria, expression of uncoupling protein mRNA in brown adipose tissue, weight gains and cumulative food intakes were measured in metformin (320 mg/kg orally for 12 days)-treated obese Zucker rats as well as in pair-fed--and in ad libitum--fed control obese rats. The weight gains were identically reduced in the metformin- and pair-fed control group compared to the ad libitum--fed rats. Metformin also significantly reduced cumulative food intake. The binding of [3H]GDP to brown adipose tissue mitochondria and the expression of uncoupling protein mRNA in brown adipose tissue were not modified by metformin. It is concluded that the weight gain reducing effect of metformin in obese Zucker rats is mainly due to reduced food intake and does not involve an effect of metformin on brown adipose tissue thermogenetic activity.

Adipose Tissue, Brown↗

PubMed2005

Screening of stimulatory effects of dietary risk factors on mouse intestinal cell kinetics.

AIM: Although epidemiological and experimental studies validate influence of genetic, environmental and dietary factors in the causation of various types of cancers including colon, results from all these sources are inconclusive. Hypothesizing that high fat diet and obesity are among the major predisposing factors in the incidence of colon cancer, we evaluated the role of diet constituted with food material derived from a tropical plant, Tamarindus indica Linn (TI). METHODS: A two part randomized double-blind study was conducted employing inbred Swiss albino mice from a single generation for the whole investigation. One day-old neonates (n = 12) were subcutaneously administered with monosodium glutamate (MSG) to induce obesity (OB). At weaning these animals were maintained on modified AIN-76 diet supplemented with 10% TI and 10% fat bolus (w/w, TIFB) for 8 wk. Subsequently, in the second part of study, four groups of animals belonging to the same generation, age and gender (n = 12 per group), were maintained on: AIN-76 control diet (CD); AIN-76 mixed with 10% TI extract (TI); and, mixed with 10% TI and 10% FB (TIFB) for 8 wk, to determine intestinal crypt cell proliferation, functionally-specific enzyme activities, fermentation profile, and energy preferences. RESULTS: We observed a significant increase in the crypt cell production rate in distal colonic segment of experimental animals when compared with the controls. This segment also contained significantly low butyrate levels compared to control and TIFB groups. All the experimental groups showed a gross decrease in the enzyme activities viz., succinate dehydrogenase, acid-galactosidase and dipeptidyl amino peptidase IV demonstrating pathological stress caused by the test regimens, and an altered metabolic flux in the cellular environment. CONCLUSION: We have demonstrated a cumulative response to the three dietary factors, one of which (TI) is reported, herein, for the first time to modulate kinetics of large intestinal mucosa, contributing to total risk posed by these test agents.

Animal Feed↗

PubMed2004

Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction.

Transgenic soybean line GTS-40-3-2, marketed under the trade name Roundup Ready (RR) soy, was developed by Monsanto (USA) to allow for the use of glyphosate, the active ingredient of the herbicide Roundup, as a weed control agent. RR soy was first approved in Canada for environmental release and for feed products in 1995 and later for food products in 1996 and is widely grown in Canada. Consumer concern issues have resulted in proposed labeling regulations in Canada for foods derived from genetically engineered crops. One requirement for labeling is the ability to detect and accurately quantify the amount of transgenic material present in foods. Two assays were evaluated. A conventional qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of soy and RR soy and a real-time PCR to quantify the amount of RR soy present in samples that tested positive in the first assay. PCR controls consisted of certified RR soy reference material, single transgenic soybeans, and a processed food sample containing a known amount of RR soy. To test real-world applicability, a number of common grocery store food items that contain soy-based products were tested. For some samples, significant differences in amplification efficiencies during the quantitative PCR assays were observed compared to the controls, resulting in potentially large errors in quantification. A correction factor was used to try to compensate for these differences.

DNA, Plant↗

PubMed2006

Phenotypic screening of hepatocyte nuclear factor (HNF) 4-gamma receptor knockout mice.

Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-gamma. HNF4-gamma is expressed in the kidneys, gut, pancreas, and testis. The first level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-gamma(+/+)), the HNF4-gamma knockout (HNF4-gamma(-/-)) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-gamma(-/-) mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.

Animals↗