Activation of tongue-expressed GPR40 and GPR120 by non caloric agonists is not sufficient to drive preference in mice.

There is mounting evidence that, in addition to texture and olfaction, taste plays a role in the detection of long chain fatty acids. Triglycerides, the main components of oils and dietary fat, are hydrolyzed in the mouth by a lingual lipase secreted from the von Ebner gland and the released free fatty acids are detected by the taste system. GPR40 and GPR120, two fatty acid responsive G-protein-coupled receptors (GPCRs), are expressed in taste bud cells, and knockout mice lacking either of those receptors have blunted taste nerve responses to and reduced preference for fatty acids. Here we investigated whether activation of those GPCRs is sufficient to elicit fat taste and preference. Five non-fatty acid agonists of GPR40 and two non-fatty acid agonists of GPR120 activated the glossopharyngeal nerve of wild-type mice but not of knockout mice lacking the cognate receptor. In human subjects, two-alternative forced choice (2-AFC) tests, triangle tests and sensory profiling showed that non fatty acid agonists of GPR40 dissolved in water are detected in sip and spit tests and elicit a taste similar to that of linoleic acid, whereas 2-AFC tests showed that two agonists of GPR120 in water are not perceived fattier than water alone. Wild-type mice did not show any preference for five agonists of GPR40, two agonists of GPR120 and mixtures of both agonists over water in two-bottle preference tests. Together these data indicate that GPR40 mediated taste perception is not sufficient to generate preference.

Murine intestinal cells expressing Trpm5 are mostly brush cells and express markers of neuronal and inflammatory cells.

To determine the role in chemosensation of intestinal solitary cells that express taste receptors and Trpm5, we carried out a microarray study of the transcriptome of FACS-sorted transgenic mouse intestinal cells expressing enhanced green fluorescent protein (eGFP) under the control of the Trpm5 promoter and compared it with that of intestinal cells that do not express eGFP. The findings of the study are: 1) Morphology and expression of markers show that most eGFP+ cells are brush cells. 2) The majority of proteins known to be involved in taste signal transduction are expressed in the eGFP+ cells, although the isoforms are not always the same. 3) eGFP+ cells express pre- and postsynaptic markers and nerves are often found in close proximity. 4) Several genes that play a role in inflammation are expressed specifically in eGFP+ cells. Furthermore, these cells express the entire biosynthesis pathway of leucotriene C4, an eicosanoid involved in modulation of intestinal smooth muscle contraction. 5) Angiotensinogen, renin, and succinate receptor genes are expressed in the eGFP+ cells, suggesting a role in the regulation of water and sodium transport, vasomotricity, and blood pressure. These data suggest that the Trpm5-expressing cells integrate many signals, including chemical signals from ingested food, and that they may regulate several physiological functions of the gastrointestinal tract.

Perception of sweet taste is important for voluntary alcohol consumption in mice.

To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: alpha-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild-type mice, whereas Tas1r3 null mice were not different from wild type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion (CTA) to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in CTA to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol.

Exercise training for patients with cardiovascular disease.

This review surveys effort training, a validated and recommended therapy, in patients with atheromatous cardiovascular disease. This true therapy reduces mortality by 25-35%, reduces clinical manifestations and complications (rhythm problems, thrombosis) and improves physical capacity, reintegration and quality of life. The effects are essentially linked to improved metabolic performance of muscles and reduced endothelial dysfunction, insulin resistance and neurohormonal abnormalities. Training also has an impact on the evolution of major risk factors, especially diabetes and arterial hypertension. The risks are limited as long as the contraindications are respected and the programmes supervised. The indications (stable angina, chronic heart failure, peripheral arterial disease) should be described more precisely by taking into account functional criteria: physical deconditioning, exclusion, compliance, mood swings, and seriousness of risk factors. The training programme should be tailor made and based on evaluation of the patient's adaptation to effort, in terms of frequency, intensity and duration of the exercises. Various types of exercise include overall or segmental physical training; concentric, eccentric, even isokinetic muscle contraction exercises; and proprioceptive rehabilitation. However, knowledge is lacking about the molecular mechanisms of the effects of training, the most effective intensity of effort, and strategies to develop physical activity in this ever-growing population for both primary and secondary prevention.

Fatigue in patients with cardiovascular disease.

Fatigue is a frequent complaint during cardiovascular disease and can sometimes constitute the first clinical manifestation of this disease. It is responsible for deterioration of the quality of life and prognosis. Although physical and mental fatigue are often intimately interrelated, these two aspects of fatigue correspond to different pathophysiological mechanisms and different clinical features and the neurobiological links between the two are only just beginning to be studied. Physical fatigue is related to loss of efficacy of the effector muscle, due to multiple causes: mismatch of cardiac output during exercise, muscle and microcirculatory deconditioning, neuroendocrine dysfunction, associated metabolic disorders. Mental fatigue corresponds to predominantly depressive mood disorders with a particular entity, vital exhaustion. The diagnostic approach is designed to eliminate other organic causes of fatigue. Functional tests investigating physical (exercise capacity) and mental dimensions (mood disorders) can be used to analyse their respective roles and to propose personalized management, in which rehabilitation has an essential place due to its global approach. The objective of this reduction of fatigue is threefold: to improve independence, to improve quality of life and to limit morbidity and mortality.

Transovarial transmission of sugarcane white leaf phytoplasma in the insect vector Matsumuratettix hiroglyphicus (Matsumura).

White leaf is a serious disease of sugarcane caused by phytoplasma. The disease is transmitted to the plant by the leafhopper Matsumuratettix hiroglyphicus (Matsumura). The reservoir of phytoplasma was suspected to be weeds that grow in sugarcane farming areas because they can be infected with phytoplasma and show symptoms similar to sugarcane white leaf. However in previous work we have demonstrated by RFLP and sequencing that this is not the case. Here we have reared M. hiroglyphicus through two generations by feeding them phytoplasma free sugarcane grown from tissue culture. By nested-PCR followed by sequencing, we demonstrated the presence of the phytoplasma in eggs, nymphs and adults of the first and second generations thereby showing transovarial transmission. We have also shown by in situ PCR that phytoplasmas were widely distributed throughout the body of the insect. RFLP and sequencing showed that the same phytoplasma was present in the vector and in the plant. Together, these data point to the leafhopper M. hiroglyphicus as the reservoir of phytoplasma that cause sugarcane white leaf disease.

Dominant loss of responsiveness to sweet and bitter compounds caused by a single mutation in alpha -gustducin.

Biochemical and genetic studies have implicated alpha-gustducin as a key component in the transduction of both bitter or sweet taste. Yet, alpha-gustducin-null mice are not completely unresponsive to bitter or sweet compounds. To gain insights into how gustducin mediates responses to bitter and sweet compounds, and to elicit the nature of the gustducin-independent pathways, we generated a dominant-negative form of alpha-gustducin and expressed it as a transgene from the alpha-gustducin promoter in both wild-type and alpha-gustducin-null mice. A single mutation, G352P, introduced into the C-terminal region of alpha-gustducin critical for receptor interaction rendered the mutant protein unresponsive to activation by taste receptor, but left its other functions intact. In control experiments, expression of wild-type alpha-gustducin as a transgene in alpha-gustducin-null mice fully restored responsiveness to bitter and sweet compounds, formally proving that the targeted deletion of the alpha-gustducin gene caused the taste deficits of the null mice. In contrast, transgenic expression of the G352P mutant did not restore responsiveness of the null mice to either bitter or sweet compounds. Furthermore, in the wild-type background, the mutant transgene inhibited endogenous alpha-gustducin's interactions with taste receptors, i.e., it acted as a dominant-negative. That the mutant transgene further diminished the residual bitter and sweet taste responsiveness of the alpha-gustducin-null mice suggests that other guanine nucleotide-binding regulatory proteins expressed in the alpha-gustducin lineage of taste cells mediate these responses.

Tas1r3, encoding a new candidate taste receptor, is allelic to the sweet responsiveness locus Sac.

The ability to taste the sweetness of carbohydrate-rich foodstuffs has a critical role in the nutritional status of humans. Although several components of bitter transduction pathways have been identified, the receptors and other sweet transduction elements remain unknown. The Sac locus in mouse, mapped to the distal end of chromosome 4 (refs. 7-9), is the major determinant of differences between sweet-sensitive and -insensitive strains of mice in their responsiveness to saccharin, sucrose and other sweeteners. To identify the human Sac locus, we searched for candidate genes within a region of approximately one million base pairs of the sequenced human genome syntenous to the region of Sac in mouse. From this search, we identified a likely candidate: T1R3, a previously unknown G protein-coupled receptor (GPCR) and the only GPCR in this region. Mouse Tas1r3 (encoding T1r3) maps to within 20,000 bp of the marker closest to Sac (ref. 9) and, like human TAS1R3, is expressed selectively in taste receptor cells. By comparing the sequence of Tas1r3 from several independently derived strains of mice, we identified a specific polymorphism that assorts between taster and non-taster strains. According to models of its structure, T1r3 from non-tasters is predicted to have an extra amino-terminal glycosylation site that, if used, would interfere with dimerization.

The molecular physiology of taste transduction.

Taste receptor cells use a variety of mechanisms to transduce chemical information into cellular signals. Seven-transmembrane-helix receptors initiate signaling cascades by coupling to G proteins, effector enzymes, second messengers and ion channels. Apical ion channels pass ions, leading to depolarizing and/or hyperpolarizing responses. New insights into the mechanisms of taste sensation have been gained from molecular cloning of the transduction elements, biochemical elucidation of the transduction pathways, and electrophysiological analysis of the function of taste cell ion channels.

Splicing variants in sheep CLN3, the gene underlying juvenile neuronal ceroid lipofuscinosis.

Mutations in different genes underlie different forms of the neuronal ceroid lipofuscinoses (NCLs, Batten disease). Subunit c of mitochondrial ATP synthase specifically accumulates in most of them, including the juvenile CLN3 form and a sheep form orthologous to CLN6. Products of these genes are likely to be components of a complex or pathway for subunit c turnover, and their expression may be cross-regulated. Different bands, some with different subcellular distributions, were detected by antisera against different regions of CLN3 on Western blots of sheep tissues. Affected liver blots were the same as controls but a specific 50-kDa band was at higher concentration in affected brain homogenates than in controls. Others have also reported bands reacting differently to different CLN3 antibodies. When the 3' end of sheep CLN3 cDNA was amplified by RT-PCR, four mRNA splicing variants were found. Different CLN3 splicing variants at the 5' end of the human cDNA have been reported. These mRNA splicing variants may account the variation of epitope distribution and the different subcellular locations of the CLN3 gene product(s). The predicted size of the unmodified CLN3 protein is 48 kDa. Significantly higher molecular weight bands may correspond to oligomers of a CLN3 isoform or to a CLN3 isoform tightly bound to another protein.

Wool production in transgenic sheep: results from first-generation adults and second-generation lambs.

In sheep transgenic for a sheep insulin-like growth factor 1 (IGF-1) cDNA driven by a mouse keratin promoter, we assessed wool production and properties in 51 adults of the first generation (G1) and in 56 lambs of the second generation (G2). Transgenic G1 sheep had an increased rate of wool production during spring and summer of year 2 compared with nontransgenic half-sibs, with a maximum increase of 17% in December, but during the winter nadir rates were similar. At second- and third-year shearing, however, fleece weights were not significantly different. There was a trend for transgenic animals to have coarser wool of lower staple strength. A controlled feeding trial revealed no significant differences in feed intake. The transgene was expressed not only in skin but also in a wide range of other tissues. Circulating IGF-1 concentrations were not significantly different between transgenic and nontransgenic animals, suggesting that local mechanisms were more important than systemic mechanisms for wool production, but were significantly higher in males than in females. In the G2 sheep, transgenic fleece weight did not differ significantly from nontransgenic either as lambs or at the end of the lamb year. Although the transgene was inherited in Mendelian fashion and was widely expressed, the production advantage seen in animals of the first generation did not persist in the second generation.

Expression of human Krev-1 gene in lungs of transgenic mice and subsequent reduction in multiplicity of ethyl carbamate-induced lung adenomas.

Mice of the A/J strain are useful models of lung cancer because they develop tumors spontaneously or after treatment with ethyl carbamate. These tumors are thought to arise from either Clara cells (papillary tumors) or alveolar type 2 cells (alveolar tumors); like many human lung adenocarcinomas, the mouse tumors involve Kiras activation. Transformation with Ki-ras can be reversed by coexpression of the Krev-1 gene in tissue culture. To test the tumor suppressor activity of Krev-1 in vivo, we produced transgenic A/J mice expressing Krev-1 under the control of the rabbit uteroglobin promoter, which directs expression of heterologous genes to the lung Clara cells. Krev-1 was expressed specifically in the lungs of transgenic mice. Sixty-six mice (35 transgenic and 31 nontransgenic) from three lines were given ethyl carbamate, and the numbers of resulting lung tumors were compared between transgenic and nontransgenic animals. The mean number (+/-standard deviation) of ethyl carbamate-induced lung tumors was 21.7 +/- 1.3 in transgenic mice and 26.9 +/- 1.3 in their nontransgenic littermates (P < 0.01). Sequencing of polymerase chain reaction-amplified ras DNA from 15 transgenic mouse tumors and 16 nontransgenic mouse tumors (controls) detected mutations in codon 61 in 13 tumors from the transgenic group and 11 tumors in the control group, whereas mutations in codon 12 were detected in only one tumor in the transgenic group and in four tumors in the controls. Together, these data demonstrate for the first time the tumor suppressor activity of Krev-1 in vivo and suggest that Krev-1 tumor suppressor activity may be specific for cells harboring mutations in codon 12 of ras.

Improved wool production in transgenic sheep expressing insulin-like growth factor 1.

Transgenic sheep were produced by pronuclear microinjection with a mouse ultra-high-sulfur keratin promoter linked to an ovine insulin-like growth factor 1 (IGF1) cDNA. Five transgenic lambs resulted from the microinjection of 591 embryos; one male and one female showed IGF1 expression in the skin. A progeny test of the ram was carried out by matings to 43 non-transgenic ewes. Of 85 lambs born, 43 (50.6%) were transgenic. At yearling shearing (approximately 14 months of age), clean fleece weight was on average 6.2% greater in transgenic animals than in their non-transgenic half-sibs, with a greater effect in males (9.2%) than females (3.4%). Transgenics showed a small but significant increase in bulk, but male transgenics had a lower staple strength than female transgenics and non-transgenics which did not differ significantly. There were no significant differences in fiber diameter, medullation, and hogget body weight. To our knowledge this is the first reported improvement in a production trait by genetic engineering of a farm animal without adverse effects on health or reproduction.

Targeting gene expression to the wool follicle in transgenic sheep.

To establish the feasibility of overexpressing foreign genes in the wool follicle, transgenic sheep were produced by pronuclear microinjection of a DNA construct consisting of a mouse ultrahigh-sulfur keratin promoter linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Four of 31 lambs born were transgenic. The overall efficiency of transgenesis was 1.1% of zygotes injected and transferred. Two transgenic rams were mated to nontransgenic ewes, and both transmitted the gene to their offspring in Mendelian fashion. CAT expression was found in the skin of one G0 ram and in 9 out of 26 transgenic G1 progeny. Two G1 lambs were sacrificed to study tissue specificity. Both had high levels of expression in skin but One had high expression in spleen and kidney with lower levels of expression in lung; the other had low expression in spleen, lung, and muscle. In situ hybridization demonstrated that transgene expression in the skin was confined to the keratogenous zone of the wool follicle cortex. Expression of CAT activity in skin was correlated with diet-induced or seasonal changes in the rate of wool growth. This keratin promoter appears useful for overexpressing factors in the wool follicle that might influence wool production or properties.

Chromosomal position effects and the modulation of transgene expression.

Chromosomal position effects can influence strongly the transcription of foreign genes in transgenic animals. This results in low frequencies and levels of gene expression and, in some cases, in aberrant patterns of expression. Strategies for overcoming these effects are described with particular reference to their application in embryonic stem cells.

A simple two-step method for efficient blunt-end ligation of DNA fragments.

The formation of recombinant plasmids results from ligation between one end of the linearized vector and one end of the insert (favored by high DNA concentration), followed by self-ligation of the newly created hybrid molecule (favored by low DNA concentration). Standard protocols recommend an average DNA concentration at which both events may occur. Since this DNA concentration is not optimum for both ligation events, efficient blunt-end ligation is compromised. We describe a method for blunt-end ligation starting at a high DNA concentration for 1 h then at 1/20 the initial DNA concentration overnight. The number of recombinant plasmids obtained with this method is about 10-fold higher than with standard protocols. Restriction digestion and agarose gel electrophoresis of 10 recombinant plasmids obtained with the two-step ligation method showed that all plasmids contained one copy of the insert.

Expression and regulation of the rabbit uteroglobin gene in transgenic mice.

The rabbit uteroglobin (UG) gene, with varying lengths of 5' flanking sequence, was introduced into the mouse genome to investigate the DNA sequences required for tissue-specific expression and regulation by steroid hormones. The pattern of expression and steroid hormone regulation of the transgene was compared to the expression and regulation of the endogenous mouse UG-like gene. In the rabbit, UG is induced in the uterus by progesterone and is expressed constitutively in the lungs, where it is weakly regulated by glucocorticoids. Genomic DNA fragments containing the complete UG-coding sequence with 4.0 (UG4.0), 3.0 (UG3.0), 2.3 (UG2.3), or 0.6 (UG0.6) kilobases of 5' flanking sequence were used to establish lines of transgenic mice. Expression of UG mRNA was observed in the lungs of UG4.0 (2/4 lines), UG3.0 (4/4 lines), UG2.3 (1/2 lines), and UG0.6 (4/4 lines) mice. Uterine expression was observed in UG3.0 (3/4 lines), UG2.3 (1/2 lines), and UG0.6 (2/4 lines). In the lungs of UG3.0 and UG2.3 mice, RNA expression was stimulated by treatment with dexamethasone. In the one line of UG3.0 mice examined, UG was regulated by ovarian steroids in the uterus. The endogenous mouse UG-like gene showed the major site of expression to be in the lung. Unlike the transgene, the endogenous gene was more strongly stimulated by glucocorticoids. Thus, we conclude that the cis elements needed for pulmonary expression of UG are contained within the UG2.3 fragment used to generate transgenic mice, but that other elements are required for full glucocorticoid regulation. Also, the transgene did not show the full uterine expression observed in the rabbit, but regulation by the ovarian steroids was observed.