Streptococcus thermophilus strains will provide a key tool to facilitate the genetic manipulation of this important dairy species.Lactic acid bacteria (LAB) are essential for the manufacture of fermented dairy products. Studies on the physiology, biochemistry and genetics of these microorganisms over the last century have contributed considerably to the improvement of fermentation processes and have resulted in better and safer products. Nevertheless, the potential of LAB is far from being maximized. The sophistication of biotechnologies and the availability of complete genome sequences have opened the door to the metabolic engineering of LAB. In this regard, the recent publication of the complete genome sequences of two  The souring of milk by microorganisms has been used for thousand of years as a natural preservation procedure . The metThe discovery and characterization of lactic acid bacteria (LAB) has tremendously modified the way fermented milk products are made. Considerable efforts have been devoted over the last fifty years to increase our knowledge about the genetics, biochemistry, and physiology of LAB. In addition to enhancing our understanding of microbial life, these studies have allowed dairy microbiologists and cheesemakers to select better strains and improve the productivity, quality, and safety of the final products. The characterization of LAB has promoted the rational development of blends of defined bacterial strains, now known as starter cultures, which are increasingly replacing the undefined blends traditionally used by the dairy industry. This strategy makes it easier to control acid production and, to some extent, phage problems. Unfortunately, it is also widely perceived that this approach reduces the much sought-after rich, complex flavour of fermented products made with undefined cultures. Thus, the search for LAB strains that produce characteristic aroma and flavours remains an attractive challenge for the dairy industry.The increasing awareness of the population of the importance of a healthy diet has led scientists to revisit the century-old hypothesis that some specific fermented dairy products may provide health benefits. A plethora of studies are underway to evaluate this concept and to improve the health benefits of LAB. Also, the long history of the safe use of these bacteria has made them very attractive candidates as vaccine delivery vehicles ,3. LastlLactococcus lactis. The publication of the complete genome sequence of L. lactis in 2001 [L. lactis via genetic manipulation [L. lactis.To meet some of these challenges and promises, it became apparent that metabolic engineering was the best approach. Owing to the industrial interest, the sound knowledge of its genetic and the availability of genetic tools, metabolic engineering of LAB has been carried out mainly with  in 2001  was a bopulation ,6 is a cpulation -12 proviStreptococcus thermophilus strains were published [S. thermophilus is a thermophilic LAB species widely used for the manufacture of yogurt and cheeses that require elevated cooking temperatures such as Swiss and Italian types. The availability of these genome sequences will make it possible to apply genomic-related analytic methods such as functional and comparative genomics, microarray technologies, proteomics, and bioinformatics to this species. These genomic-based technologies will undoubtedly accelerate the metabolic engineering of S. thermophilus, which is still at its infancy. We will briefly discuss current studies that will greatly benefit from these S. thermophilus genomic sequences: food-grade vectors, optimization of lactose metabolism and exopolysaccharide (EPS) production, and resistance to bacteriophages.Recently, the complete genome sequence of two ublished  and a thublished . S. therS. thermophilus strains[Metabolic engineering often involves inactivation or overexpression of relevant chromosomal genes or stringent control of an extrachromosomal foreign gene borne by a plasmid artificially introduced into the host. Plasmids thus play a central role in these studies because they are the primary vehicles used to manipulate target DNA sequences. However, these extrachromosomal genetic elements are naturally but infrequently observed in s strains.S. thermophilus, an efficient, stable, food grade expression system is still lacking. The recent discovery of a novel theta-type replicating plasmid [S. thermophilus, such as galactokinase [S. thermophilus genome sequences also provide crucial information on transcription and translation initiation signals and the codon usage of this bacterium. This may help in the design of efficient expression systems.Understandably, very few cloning tools are at hand, and those that are available are based on similar replication machinery. This lack of diversity poses significant problems for many genetic studies. While several types of transformation methods have been developed to introduce foreign DNA into  plasmid  as well tokinase  and alphtokinase , suggestS. thermophilus to rapidly take up and metabolize lactose is crucial in several fermentation processes. While S. thermophilus readily metabolizes the glucose moiety of lactose, it is unable to metabolize galactose, which is expelled into the external medium. The presence of galactose in yogurt and other dairy products might be unwanted for different reasons. Notably, galactose is poorly metabolized by humans and may cause, under certain conditions, health problems [S. thermophilus results either from poor expression of the gal operon [galK gene coding for galactokinase [galK translation was first suggested by a comparative analysis of the S. thermophilus gal-lac operons with the homologous genes from the phylogenetically related oral bacterium Streptococcus salivarius. This is a convincing example of how comparative genome analysis may help to decipher specific metabolic pathways of LAB. While the introduction of a functional extrachromosomal S. salivarius galK allele in S. thermophilus allows it to grow on galactose, it does not prevent the expulsion of galactose during growth on lactose [S. thermophilus genes are not functional (pseudogenes) and that one third of these pseudogenes are dedicated to sugar metabolism [S. salivarius genome would be welcome since many S. thermophilus pseudogenes involved in sugar transport are functional in S. salivarius [The ability of problems ,19. The l operon  or inefftokinase ,21. The  lactose . In thistabolism . It woullivarius .S. thermophilus are widely used for the commercial manufacture of yogurt because they produce exopolysaccharides (EPS), which give a viscous texture to the fermented dairy product. Other EPS-producing S. thermophilus strains can also enhance the functional properties of some cheeses such as Mozzarella. The organoleptic properties of these products are largely due to the amount and types of exopolysaccharide produced during the fermentation process. The yield and sugar constituents of EPS are influenced by several factors and vary from strain to strain [S. thermophilus does not naturally produce large amounts of EPS, which explains why considerable efforts are being directed toward understanding the cellular mechanisms of EPS biosynthesis. Since low levels of sugar precursors may limit EPS synthesis, a better understanding of S. thermophilus metabolic flux during sugar metabolism may lead to new strategies to enhance EPS production [S. thermophilus by metabolic engineering of central carbon metabolism. Moreover, because EPS yields are growth associated, efforts to increase production levels are likely to require novel strategies to enhance biomass production. This task clearly requires a comprehensive view of the cell machinery. The sequencing of a whole genome is a mandatory step to achieve this goal. As our knowledge of S. thermophilus continues to improve, novel EPS and/or applications for EPS+ cultures are likely to emerge.Some strains of o strain . S. theroduction . Levandeoduction  showed tS. thermophilus hosts should provide new insights in several areas of phage research.Bacteriophages are the most significant cause of fermentation failures in the dairy industry worldwide. Dairy microbiologists have attempted for more than 70 years to eliminate, or at least bring under better control, the bacteriophages that interfere with the manufacture of many fermented milk products . The pubS. thermophilus mutants is generally the first approach used to transform a phage-sensitive strain into a phage-resistant mutant, most likely following spontaneous chromosomal mutation in the gene encoding the phage receptor. Although progress has been made in identifying phage proteins involved in S. thermophilus host recognition [The development of bacteriophage-insensitive ognition , the ideognition , a numbeS. thermophilus phage genomes are now available [S. thermophilus phages are already available[At least eight complete vailable ,28. Phagavailable, meaningS. thermophilus genome sequences will obviously facilitate our understanding of the metabolic potential of S. thermophilus. It will also make it easier to design rational genetic manipulations of this important dairy bacterium in order to produce added value cheeses and yogurts and to use it as a cell factory. In addition, knowing the complete genome sequences should lead to the development of new genetic tools that will provide insights into the evolution of microbial communities, shifts in metabolism, and how each member adapts to the environmental changes that occur during complex fermentation processes.A thorough understanding of LAB metabolism and how it is regulated by external stimuli is a prerequisite for maximizing the potential of LAB. The availability of complete S. thermophilus genome sequence have opened up exciting, new possibilities that will build on an already rich heritage.The exciting biotechnological developments in LAB and studies that are already underway will benefit both consumers and the dairy industry. The availability of the complete


There are likely many scenarios and pathways that can lead to metabolic syndrome. This paper reviews mechanisms by which the accumulation of visceral adipose tissue (VAT) may contribute to the metabolic syndrome, and explores the paradigm of a critical VAT threshold (CVATT). Exceeding the CVATT may result in a number of metabolic disturbances such as insulin resistance to glucose uptake by cells. Metabolic profiles of patients with visceral obesity may substantially improve after only modest weight loss. This could reflect a significant reduction in the amount of VAT relative to peripheral or subcutaneous fat depots, thereby maintaining VAT below the CVATT. The CVATT may be unique for each individual. This may help explain the phenomena of apparently lean individuals with metabolic syndrome, the so-called metabolically normal weight (MONW), as well as the obese with normal metabolic profiles, i.e., metabolically normal obese (MNO), and those who are "fit and fat." The concept of CVATT may have implications for prevention and treatment of metabolic syndrome, which may include controlling dietary carbohydrates. The identification of the CVATT is admittedly difficult and its anatomical boundaries are not well-defined. Thus, the CVATT will continue to be a work in progress. Arguably, the major pathogenic factor in the metabolic syndrome is central obesity ,2. WhileTo date, it has not yet been established that insulin resistance, i.e., resistance of cells to insulin's effects, is responsible for the onset of the multiple risk factors associated with insulin resistance syndrome and subsequent development of atherosclerosis and cardiac events . In factThe term "metabolic syndrome" is now preferable to "insulin resistance syndrome," and has a prevalence of 25 percent in U.S. individuals age >20, rising to >40 percent by age 60 . The impectopic fat storage syndrome hypothesis suggests that as adipocytes hypertrophy and reach their capacity for storing more fat, then additional fat from excess dietary lipids or calories is deferred to non-adipose tissues intracellularly, e.g. liver, skeletal muscle, heart, and the beta cells of the pancreas where they can exert toxic effects and dysfunction [The function . This "lfunction ,28-30. Ffunction . Comparefunction ,33.It must be emphasized that the current proposal is a working hypothesis. Figure It is encouraging that only a modest loss of 5–10 percent of body weight in obese patients is associated with preferential mobilization of VAT compared to SCAT, leading to simultaneous improvement in all metabolic markers of CHD risk. Such modest weight loss can prevent and reverse type 2 diabetes -48, and With total weight loss, those with greater amounts of VAT initially lose more VAT, and VAT is more sensitive to weight reduction because the VAT adipocyte is more metabolically active and sensitive to lipolysis ,54. AfteThe identification of the CVATT is admittedly difficult and its anatomical boundaries are not well-defined. Thus, the CVATT will continue to be a work in progress. While there are numerous studies linking VAT quantity to insulin resistance and metabolic syndrome, this does not necessarily prove that VAT is the cause. However, there are a number of plausible mechanisms linking VAT to the metabolic syndrome.Once thought to be an inert energy storage depot, adipose tissue is now known to be a critical endocrine organ. The term "adipocytokines" or "adipokines" has been used to describe the numerous adipocyte secretory products which include: adiponectin, adipsin, estrogen, angiotensin II, angiotensinogen, leptin, plasminogen activator I (PAI-1), agouti protein, resistin , acylatiAdiponectin has many beneficial vascular and metabolic effects, e.g., it serves as an anti-inflammatory molecule for vascular walls as well as adipose tissue, inhibits vascular smooth muscle proliferation, protects endothelium from macrophage adhesion and macrophage-induced injury , may incIronically, although produced by adipose tissue, adiponectin levels are lowered with greater degrees of obesity and with overfeeding. Decreased concentrations of adiponectin are associated with type 2 diabetes, hypertension, elevated glucose levels, insulin and TGs, and cardiovascular disease (CVD). It has been suggested that adiponectin is under feedback inhibition in obesity and reduced in patients with metabolic syndrome . AdiponeFat mass can expand in one of two ways: individual adipocytes can increase in volume or they can increase in number as more are derived from preadipocytes. As adipocytes grow larger, they become dysfunctional. The total number of adipocytes is increased with increasing fat mass, but it is the increased number and percentage of large adipocytes, compared to the smaller ones, that may partially account for the inability of adipose tissue to function properly . While tPreadipocytes from the SCAT depots have a greater differentiation capacity than those from the VAT depots ,71. The TZDs can increase the number of new fat cells, and because obesity is a major cause of insulin resistance, this represents an apparent paradox. Ex-vivo studies of human preadipocytes from SCAT and VAT depots have demonstrated that TZD-stimulated differentiation is much greater in SCAT than VAT preadipocytes . Since TMacrophages increase their accumulation within fat depots in direct proportion to increases in adipose tissue and adipocyte size. The increased macrophage activity observed in the adipose tissue of the obese may reflect a combination of conversion of local preadipocytes to macrophages and activation and recruitment of resident macrophages and circulating monocytes. This seems to occur after the onset of adiposity but prior to insulin resistance, and supports the notion that pathophysiological consequences of obesity involve macrophages and inflammation that contribute to insulin resistance and metabolic syndrome ,79. EvidThere are numerous inherent differences between VAT and SCAT. VAT is a major predictor for insulin resistance  and metaRecently, omental VAT cells have been shown to have an approximately two-fold higher rate of insulin-stimulated glucose uptake compared with SCAT adipocytes, and this could be explained by a higher GLUT-4 expression . PerhapsVAT has a high density of androgen receptors and testosterone which can amplify its own effect by up-regulation of androgen receptors, inhibiting the expression of lipoprotein lipase (LPL) and FA uptake ,88. In mPatients with type 2 diabetes and metabolic syndrome often appear Cushingoid, yet they invariably do not have elevated plasma cortisol . CompareThe amount of fat deposited within skeletal muscle (intramyocellular lipid – IMCL) and the ability of muscle to oxidize fat are important determinants of weight gain, weight r2 and BMIs from 19 to 39 kg/m2 [Colberg et al studied the fasting patterns of skeletal muscle fatty acid uptake and oxidation in healthy, lean and obese premenopausal women who had a cross-sectional VAT area over a range from 18 to 180 cm39 kg/m2 . The res39 kg/m2 . TogetheMauriege et al found that adrenoreceptor sensitivity was increased in SCAT cells of individuals who have a higher VAT accumulation compared to those with a low VAT deposition . SCAT adThe PPARs are important transcription factors that play an important role in the induction of adipose-specific genes, the proliferation and differentiation of adipocytes, and the development of mature adipose tissue. A number of transcription factors are involved, including PPARγs. Giusti et al suggest that in VAT, the expression of PPARγ2 is controlled by local transcription factors  promoting fat storage in adipocytes. Given that the fat storage capacity is limited in VAT, RXRα induces the expression of PPARγ2 in SCAT to increase its overall capacity . These dPerhaps the above can be explained as follows. SCAT cells may act as a buffer or sink for circulating FAs and TGs but once they reach their capacity they lose their protective benefits. Initially, VAT may influence SCAT to expand and act as a buffer. However, once the critical VAT threshold (CVATT) is achieved and metabolic syndrome has begun to develop, then VAT may influence central SCAT to become more VAT-like, i.e., more lipolytic and less sensitive to insulin's adipogenic or lipid storing effects.As discussed earlier, preadipocytes from SCAT depots have a greater capacity than VAT to differentiate into numerous, small, insulin-sensitive, adipocytes ,71. ThesIn defending the role of VAT accumulation in individuals with metabolic syndrome, we must postulate a high rate of lipid turnover, with high rates of lipolysis at certain times matched by high rates of lipid deposition at other times. Otherwise, as Frayn points out, the hyperlipolytic VAT would ultimately disappear . He alsoWhile some studies cast doubt on the portal theory and its implications for VAT's direct delivery of FFA to the liver ,119, theIf trunk fat is taken into account, accumulation of fat in the hips and legs is an independent predictor of lower cardiovascular and diabetes-related mortality, and it seems to protect against impaired glucose metabolism, especially in women -124. In When there is an inability to store fat, due to lipodystrophy, the adipocytes' storage capacity is exceeded and lipids accumulate and cause lipotoxicity in liver, muscle, and other organ tissues . A countEstrogen promotes the accumulation of peripheral gluteo-femoral SCAT, which may be protective . The abuAfter menopause, when ovarian function declines, adipocytes become the primary source of endogenous estrogens , and comAlong with an increase in VAT, a decline in estrogen is also associated with reduced lean body mass as well as other features of the metabolic syndrome including: dyslipidemia with elevation in Lp(a), triglycerides, and an increase in small, dense, LDL particles. Estrogen deficiency also may influence cardiac risk by its effects on the insulin action, the arterial wall, and fibrinolysis. Park et al showed that postmenopausal women lost less VAT compared with the premenopausal women during a weight reduction program (10.5 percent vs. 25.7 percent respectively) . The reaAs mentioned above, in menopause, adipocytes are primary sources of endogenous estrogens in women ,134, andCompared to men of similar age, premenopausal women appear to be significantly protected from CHD. However, by age 70 the incidence of CHD is equal in men and women, suggesting that estrogen deficiency causes a rapid acceleration in CHD risk . Yet, inObesity, particularly visceral obesity, as well as insulin resistance and hyperinsulinemia are associated with breast cancer . InsulinAround 1900, most women died soon after menopause. The average lifespan of persons in the United States has since lengthened by greater than 30 years , which mWhen one consumes too many calories, especially in the form of excessive carbohydrates, the liver converts excess glucose to fatty acids. First, glucose that is not oxidized or stored as glycogen is metabolized to acetyl CoA, which then enters the lipogenic pathway. Acetyl CoA is catalyzed to form malonyl CoA, which in turn inhibits carnitine palmitoyl transferase 1  . The netThe first adipocyte-specific hormone to be characterized, leptin is produced predominantly by SCAT adipocytes compared to VAT. Females produce leptin at about twice the rate in males , and lepThe increase in leptin concentration after meals is not simply a result of a caloric load, but is in response to a signal that is not present following a fat load without carbohydrate . Leptin Excess caloric consumption and fat deposition results in newly synthesized FAs that are transported as VLDLs and stored as TG in adipocytes. Initially, these expanding adipocytes secrete leptin in proportion to their growing fat accumulation. Leptin also crosses the blood brain barrier, stimulates its receptor in the hypothalamus, and causes the release of neuropeptide-Y (NP-Y), which reduces feeding behavior . This, iAs the process progresses, inefficient leptin action can lead to the opposite of leptin's protective effects, e.g., hyperphagia, decreased fat oxidation, increased tissue TG levels, insulin resistance, and overweight. Subsequently, plasma leptin levels rise. The majority of obese individuals with high leptin levels show a leptin insensitivity or "resistance ," which It is quite possible that hyperleptinemia in diet-induced obesity serves to protect nonadipose tissues  from the toxic effects resulting from the spillover of full adipose stores and subsequent ectopic deposition of FFAs. In defense of this paradigm, Unger points out that normally rats can tolerate a 60 percent fat diet because 96 percent of the surplus fat is stored in an enlarging adipose tissue mass, in which leptin gene expression increases proportionally . HoweverAcquired leptin resistance occurs in aging, obesity, Cushing's syndrome, and acquired lipodystrophy, a condition associated with protease inhibitor therapy of AIDS. Preliminary evidence suggests that patients with these conditions have increased ectopic fat, i.e., lipid deposition in non-adipose tissues .The relation between cerebrospinal fluid and serum levels of leptin in obese humans suggests that defective blood brain barrier (BBB) transport accounts for a great deal of leptin resistance in the CNS. Banks et al showed in mice that serum TGs directly inhibit the transport of leptin across the BBB and so could be a major cause of leptin resistance across the central nervous system (CNS). Thus they suggest that serum TGs are likely a major cause of the leptin resistance seen in both obesity and starvation . This hyCompared to VAT, SCAT is the predominant source of leptin , yet patA number of clinical states exhibit evidence of leptin insufficiency, either leptin deficiency or resistance, and they all have in common the metabolic syndrome. These include rare genetic diseases known as lipodystrophies, which are characterized by a redistribution of fat. Ironically, in the more severe cases, e.g., congenital generalized lipoatrophy, near-complete fat loss is associated with severe insulin resistance, fatty liver, and classic features of the metabolic syndrome. There is hyperleptinemia along with hyperphagia and a predominance of intra-muscular fat . DunnigaThe aforementioned potential role of TGs in leptin resistance may have implications for patients with lipodystrophy and lipoatrophy who have little or no fat mass, and as a result, have very little or no leptin. They also have severe hypertriglyceridemia that is reversed by treatment with leptin ,171. TheTransplantation of adipose tissue grafts in animal models of congenital lipoatrophy reverses the signs of the metabolic syndrome in a dose-dependent fashion . FurtherLike leptin, adiponectin secretion increases early on in obesity and plays a role in reducing the expression of lipogenic enzymes and increases FA oxidation in peripheral tissues thus limiting ectopic fat accumulation . The facThe CVATT has tremendous individual variation; thus a relatively "thin" individual  and an excess of VAT for him, may be metabolically obese, normal weight (MONW) . Meanwhi2 was associated with an increased risk of CHD in pre-and postmenopausal women [2 was associated with increased CHD risk in young adult men and premenopausal women (mostly of French Canadian descent) [2 has also been observed by others [2 for metabolic deterioration [2 for men and > 90 cm2 for women [A number of studies have looked at a possible CVATT -182. Usial women . Similardescent) , and a cy others ,181. Othioration ,184. De ioration . As discor women .2 would show greater improvement in their metabolic profile compared to those who also lost weight but remained above the 110 cm2 VAT threshold [2 [2 CVATT but their longitudinal data from both pre- and posttreatment suggest that these women should reduce their VAT area to <60 cm2 through weight reduction to improve CHD risk factors [Brochu et al were unable to demonstrate that obese postmenopausal women who reduced their weight and attained a level of VAT below 110 cmhreshold . Howevershold [2 . It shoushold [2 ,121,125. factors .2 by abdominal CT), insulin resistance, and hyperinsulinemia [VAT accumulation contributes to metabolic risk factors in nonobese individuals ,188. Rudulinemia -26. As pulinemia -41,191. ulinemia ,193.Both low birthweight (LBW)  and lowe2) fulfill the criteria for MONW [One should consider ethnic differences when attempting to identify MONW subjects. Lean appearing individuals, especially in certain ethnic groups such as the Japanese, may have significant amounts of VAT that surpass their CVATT but appear with what, for the general population, would be considered a normal BMI and waist circumference . For exafor MONW ,181. In for MONW . This ilfor MONW . On the for MONW .McGarry found that one of his most obese patients in a series (BMI 32.8 kg/m2) was one of the most insulin-sensitive but had one of the lowest values for intramyocellular lipid (IMCL). Conversely, another subject, with a BMI of only 18.9 kg/m2, proved to be highly insulin-resistant but had a large amount of IMCL. This supports that insulin sensitivity appears to correlate more with where the fat is located rather than the total amount in the body . This haLike some of the Micronesian Nauruans and Indian-Fijians above, there are individuals who are obese and who nevertheless are metabolically normal – "metabolically normal obese; MNO." Unlike their MONW counterparts, MNO individuals have very little VAT accumulation. They often share an onset of obesity early in childhood, normal VAT, lower TGs, and increased HDL. The actively competitive Japanese wrestlers maintain their gross obesity by consuming a 5,000 to 6,000 calorie diet. They are MNO, and their VAT is normal in amount, i.e., they have excessive amounts of SCAT . On retiSeveral investigators have found that there has been a positive association between insulin sensitivity and duration of obesity, i.e., those who are obese since childhood are more likely to remain insulin sensitive. In one study 48 percent of the MNO women presented with a history of an earlier age-related onset of obesity (between 13 and 19 years of age) and less VAT compared with 29 percent of the metabolically abnormal obese .Insulin sensitivity seems to be dependent upon adipose cell size; as adipocytes within tissue grow larger, they become more insulin resistant . Normal-During puberty, a certain degree of insulin resistance is normal, and children who are more insulin resistant have decreased SCAT fat gain . Early iVAT is strongly associated with fitness even within individuals of the same weight. This is illustrated by the earlier mentioned example of the active Sumo wrestler in his prime who has relatively little VAT . RegularIn obese patients, increasing physical activity can enhance fat oxidation, reduce IMCL and improve insulin sensitivity . ExercisRoss et al showed that either modality, caloric restriction alone or daily exercise without calorie restriction, is an effective strategy for reducing obesity in moderately obese men.2) and was dose-dependent. The women who exercised for approximately 200 min/wk lost 4.2 percent of total body fat and 6.9 percent of VAT without reducing their energy intake [Their findings also suggest that exercise without weight loss is a useful method for reducing VAT and preventing further increases in obesity . Irwin ey intake . Exercisy intake .While evidence supports that CRF may be associated with a lower VAT, this is certainly not proven. However, study results suggest that individuals with moderate to high CRF levels have lower WC than men with low CRF independent of BMI ,222. DatSurgical removal of VAT in animals and humans dramatically improves insulin resistance and diabetes. In a Swedish, single-center, randomized and controlled pilot trial of 50 severely obese adults, Thorne et al compared 25 patients who underwent adjustable gastric banding (AGB) alone with AGB plus surgical removal of the total greater omentum. At two-year follow-up there were no statistical differences between groups with regard to weight loss, changes in WHR or sagittal diameter. However, the improvements in oral glucose tolerance insulin sensitivity and fasting plasma glucose and insulin were 2–3 times greater in omentectomized as compared to control subjects, which was statistically independent of the loss in BMI . More reRecently Klein et al. demonstrated that liposuction conferred no benefits with regard to metabolic profile . FurtherSince our genes have not changed significantly in the past 10,000 years, the rise in obesity can be attributed to the environment, including what we are exposed to in the way of food as well as the level of physical activity. While the main focus has been on diet and activity, what may be overlooked is the tremendous increase in exposure to synthetic organic and inorganic chemicals, which can damage many of the mechanisms involved in weight control. Most of us have been exposed to organochlorines found in pesticides, dyes, solvents, etc., and we contain residues in our adipose tissue, where they are preferentially stored. Thus, the obese tend to have increased organochlorine concentrations compared to lean individuals . During Frisancho points out that an important contributing factor for obesity in modern as well as developing nations is a reduced fat oxidation and increased metabolism of carbohydrate. This has been brought about by a shift toward the body's preference toward oxidizing carbohydrate rather than fat – resulting in an increased deposition of body fat. In developing nations, obesity can co-exist with developmental undernutrition, which can result in obesity with short stature .A solution to reducing the ectopic fat, as well as VAT, burden would be to enhance its oxidation in nonadipose tissues, e.g., liver, pancreas, and skeletal muscle. This will push the system toward below the CVATT and improve insulin sensitivity. In their review, Westman et al cite many studies that have consistently shown that low-carbohydrate/high-fat diets consumed for more than seven days induce powerful metabolic adaptations to enhance fat oxidation . Such diOptimizing macronutrients and food preparation can have beneficial effects in those with visceral obesity. A number of recent reviews support the metabolic benefits of controlling glycemic index (GI)  and glycVAT cells have a two-fold higher glucose uptake rate compared with SCAT cells . It may A number of studies have demonstrated an association between glycemic load (GL) and levels of CRP ,245, whiCompared to SCAT, VAT (both adipose and non-adipose cells within VAT) is associated more with PAI-1 – a powerful risk factor for CHD ,252. In Esposito et al demonstrated in both diabetics and non-diabetics that after consuming a high carbohydrate high-fiber meal, IL-18 (a potent pro-inflammatory cytokine) concentrations increased . AdiponeRecently, dietary TGs have been demonstrated to contribute to CNS leptin resistance by impairing the transport of leptin across the blood brain barrier where it would usually stimulate the release of neuropeptide-Y and reduce feeding behavior . ReducinLeptin may enhance fatty acid oxidation and protects against fat deposition and lipotoxicity. As mentioned earlier, normally, rats can tolerate a 60 percent fat diet because 96 percent of the surplus fat is stored in an enlarging adipose tissue mass, in which leptin gene expression increases proportionally . HoweverAcute overfeeding can cause circulating leptin levels to rise by 40 percent and more than three-fold after chronic overfeeding, whereas fasting is associated with a decreased leptin levels. This may suggest that overfeeding leads to leptin resistance. Dietary carbohydrates may influence leptin action. Some investigators have suggested that the increase in plasma leptin concentration observed after meals is not simply a result of an energy load but is in response to a signal that is not present following a fat load without carbohydrate . SCAT-deThere is an association with lifestyle, worry, cortisol levels, and abdominal girth. Those who were found to have the highest levels of chronic stress had the highest levels of cortisol and VAT -257. ThiThere are many other ways in which psychological stress might increase the likelihood of developing metabolic syndrome and type 2 diabetes, for example, chronic psychological stress may also be related to central activation of the HPA  axis and the sympathetic nervous system (SNS) . PsycholStress decreases splanchnic blood flow, impairs the integrity of the GI tract, increases intestinal permeability, and results in increased absorption of lipopolysaccharide endotoxin (LPS) from the gut (the greatest source of LPS). Elevated portal bloodstream LPS levels stimulate Kupffer cell receptors and cytokine release and possibly other immune-challenging activators, e.g., AGEs in food .Dietary carbohydrate has been known to stimulate SNS activity though a number of studies have emphasized the role of insulin. Recent studies in rats have demonstrated that adding glucose to the basic diet increased SNS activity in peripheral tissues and increased GLUT 4 activity in interscapular brown adipose tissue and retroperitoneal fat  . OverfeeChronic stress leads to elevated cortisol levels, which may lead to accumulation of VAT and metabolic syndrome . Stress-The role of visceral adipose tissue (VAT) obesity in metabolic syndrome is critical and complex. (See figure The author(s) declare that they have no competing interests.


To assess the accuracy of Dual-energy X-ray absorptiometry (DXA) in underweight patients with chronic gastrointestinal disease, we investigated the ability of DXA to detect variations in body composition induced by infusion of parenteral nutrition (PN). Furthermore, the influence of a low body weight per se on the accuracy of DXA was studied by placing packets of lard on lean healthy subjects.2. The lard study, where packets of lard were placed either over the thighs or the trunk region, was performed in 8 healthy lean male volunteers, 26.4 ± 7.4 yr., and 21.0 + 0.9 kg/m2. Body composition, including measures of the total mass (TM), soft tissue mass (STM), lean tissue mass (LTM), fat mass (FM), and total body mineral content (TBBMC), was assessed by DXA. The fat fraction of the lard packets (3.49 kg), measured in triplicate by chemical fat extraction, was 52.2%.The hydration study included 11 patients with short bowel syndrome on long-term home parenteral nutrition (9 women and 2 men), and (mean ± SD) 49.5 ± 17.1 yr., 19.3 ± 3.1 kg/mHydration study; The increase in scale weight (BW) of approximately 0.90 kg due to infusion of PN correlated significantly to the increase in TM , and the increase in STM , however not with the increase LTM . Mean changes in TM (0.88 kg), STM (0.88 kg), and LTM (0.81 kg) were not significantly different from changes in BW (p > 0.05). Lard study; Regardless of position, measurements of FM and LTM of the added lard were not significantly different from expected values. However, the composition of the lard packets into FM and LTM was more accurately detected when the packets were placed over the thighs than over the trunk region. The accuracy of DXA in individual subjects, expressed as the SD of the difference between expected and measured values, was 1.03 kg and 1.06 kg for the detection of changes in LTM and FM, respectively, and 0.18 kg for the detection of changes in STM and TM.On a group level, DXA provided sufficient accuracy to detect small changes in body composition in underweight patients with chronic gastrointestinal disease. However, the accuracy errors were higher than reported in normal weight subjects. The accuracy was not influenced by a low body weight per se. Malnutrition is commonly observed in patients with chronic gastrointestinal disease and nutritional support is therefore an integral part of the management of these patients. Accurate and precise methods that are sensitive enough to track small changes in body composition are important for assessing the effect of nutritional intervention. At present, there are available a variety of well-established techniques such as hydrodensitometry, isotope dilution, air-displacement plethysmography , and potDual-energy X-ray absorptiometry (DXA) is a precise, accurate, non-invasive, safe, and convenient technique, founded on a three compartment model separating the body into total body mineral mass (TBBMC), FM, and lean tissue mass (LTM), the latter being the remaining bone-free fat-free tissue mass ,3. In thThe purpose of this study was twofold. Primarily, in underweight patients with short bowel syndrome dependent on long term home parenteral nutrition we evaluated whether DXA could accurately detect small changes in body composition induced by intravenous infusion of parenteral nutrition (PN). Secondly, to explore the influence of a low body weight per se on the accuracy of DXA, we investigated the ability of DXA to detect changes in body composition by placing packets of lard on the trunk and thighs of lean healthy volunteers.Two separate experiments were undertaken. In the hydration study, changes in body composition were induced by intravenous infusion of PN in underweight patients with short bowel syndrome. In the lard study, changes were induced by placing packets of lard on lean healthy volunteers.2).All participants were weighed (after voiding) on a calibrated digital scale accurate within 0.1 kg (subjects wearing light clothes). The heights were measured after a maximal inhalation to the nearest 0.1 cm by using a wall-mounted stadiometer. The averages of two measurements for both height and weight were used as the criterion measurement. The body mass index was calculated as weight divided by height squared  with the subject supine. The host software was rev. 2.5.2. and the scanner software rev. 2.0.0. The theory and methodology for body composition by DXA has previously been described [et al.  have rep2). The diagnoses were: Crohn's disease (n = 6), ischaemic infarction (n = 2) and other (n = 3). Patients were on average (mean ± SD) 49.5 ± 17.1 yr., 1.58 ± 0.07 m, 48.5 ± 9.5 kg, and 19.3 ± 3.1 kg/m2. The PN was in all patients provided as a 3 L 'all in one' plastic bag containing a fixed composition of protein, glucose, and electrolytes, and four patients had additional supplements of 1–2 L of saline. The infusion was given continuously over a period of 8–10 hours during the night. Before starting the infusion all patients were weighed on a scale and scanned as described below. Immediately after completing the infusion the patients were reweighed and rescanned. Due to the large volume of intravenous fluid provided with the parenteral nutrition, patients were allowed to void during the study.This study comprised 11 patients (9 women and 2 men) with short bowel syndrome treated with daily supplements of PN. The participants were selected for low body weight (BMI < 22 kg/mST) of an 'all in one' 3 L bag of PN was calculated using the equation RST = Σ (-fi × (μmi)L) / Σ (-fi × (μmi)H), where (fi) is the mass fraction, (μmi) is the mass attenuation coefficient of the i'th component at high (H) and low (L) photon energy levels [ST-value for PN was calculated to 1.365, which is very close to that of normal saline (1.377). Given the calculated RST-value PN should theoretically be scanned by DXA as consisting of approximately 2% FM and 98% LTM. Such values were confirmed in vivo by scanning one subject three times before and after placing 2 bags (6.96 kg) of PN on the subject's legs. By DXA, the equivalent 6.99 kg increase in TM (TM) resulted from a 7.31 kg gain of LTM (104.5%) and a 0.32 kg loss of FM (- 4.6%), whereas the TBBMC changed only 6 g (0.1%).The theoretical soft-tissue attenuation . Eight healthy lean male volunteers, who were on average (mean ± SD) 26.4 ± 7.4 years of age, agreed to participate. Anthropometric measures of the participants were taken immediately before the study and averaged 1.81 ± 0.07 m in height, 69.0 ± 7.7 kg in weight, and 21.0 ± 0.9 kg/m2 in BMI. Without reposition, four consecutive total body DXA scans were performed on each participant. Two scans without added lard served as baseline measurements , and two scans were performed with the lard packets placed alternately on the abdomen centred over the lumbar vertebrae, and on the thighs at midpoint of the femur. The placements of the lard over the thighs and trunk were chosen to represent regions where the ability of DXA to correctly measure soft tissue composition is known to be good and poor, respectively.For this experiment two packets of porcine lard  were constructed and enclosed in plastic wrap. The lard packet dimensions were 19.8 cm × 38.8 cm × 2.6 cm, and weighed, using a beam scale, 3.49 kg. The total fat fraction of the lard, measured in triplicate by chemical fat extraction according to the method of Folch et al. , was 52.The Ethics Committee for Medical Research in Copenhagen, Denmark, approved the study protocol and the study was conducted in accordance with the Declaration of Helsinki of 1975, as revised in 1983. Written and oral informed consent was obtained from all patients prior to inclusion.p value of less than 0.05 was considered statistically significant. The SPSS statistical program version 10.0  was used for all analyses.All results are expressed as means ± standard deviation (SD) unless otherwise indicated. A paired Students t-test was used to compare paired variables. Association between variables was established by Pearson's correlation coefficients and linear regression. The CV%'s for the measurements of TBBMC or FM were calculated from the within-subjects SD's divided by their respective grand means. All statistical tests were two-tailed, and a The descriptive statistics for body weight (BW) and body composition variables by DXA before and after intravenous infusion of PN are given in Table The placement of 3.49 kg of exogenous lard (with a composition of 52.2% FM and 47.8% LTM) over central and peripheral regions of the body of eight lean healthy volunteers had no effect on the estimate of TBBMC . The descriptive statistics for the corresponding mean changes in TM, STM, LTM, and FM are given in Table Compared to several other body composition techniques DXA has a very high precision ,11, whicIn addition to a high precision, DXA has proven an accurate method for body composition analysis in normal weight healthy subjects -17. Howeet al. [et al. [We studied underweight patients with gut failure due to short bowel syndrome, who were dependent on long-term home parenteral nutrition. The changes in TM, STM, and LTM of 0.90 kg induced by infusion of PN were accurately detected on a group level. Furthermore, measurements of TBBMC and FM, body constituents that should not be affected by changes in hydration, remained unchanged during the experiment, and the respective CV%'s were very close to normal precision errors for repeated measurements ,8,11. Ouet al. . Yet, in [et al. , a diffe2) with packets of lard placed over the trunk and thigh region. In agreement with previous studies in normal weight healthy volunteers [2) per se does not affect the accuracy of DXA. DXA appeared somewhat more accurate in detecting both the mass and composition of the added lard when the packets were placed over the thighs. Thus, the FM was slightly underestimated and the LTM correspondingly overestimated when the lard was placed over the trunk. Limitations in the ability of DXA to accurately assess the composition of soft tissue in the trunk region have been reported earlier. Thus, in common with our results Snead et al. [et al. [To explore the influence of a low body weight per se on the accuracy of DXA, we investigated otherwise healthy lean young men  densitometer to measure small changes in soft tissue composition in underweight patients with chronic gastrointestinal disease. DXA accurately detected changes in TM, STM, and LTM induced by infusion of PN on a group level, however the accuracy errors were up to 100% higher in this group compared to normal weight healthy subjects. Also, DXA performed well in detecting the composition of added lard placed on lean healthy subjects with accuracy errors similar to those reported in normal weight subjects, supporting that a low body weight per se does not affect the accuracy of DXA.We conclude that DXA is an accurate method for body composition analysis in underweight patients with chronic gastrointestinal disease. The individual accuracy errors however were higher than in normal weight subjects and this should be taken into account when evaluating the changes in body composition in the individual patient.KH and MS were responsible for conception and design of the study. KH, PHH and MS were responsible for data interpretation, and manuscript preparation. None of the authors have personal or financial interests in any organization sponsoring the research.


Caenorhabditis briggsae, strain-specific variation in ray pattern has provided an entrée into the evolution of ray pattern. Some strains were fixed for a derived pattern. Other strains were more plastic and exhibited derived and ancestral patterns at equal frequencies.How does intraspecific variation relate to macroevolutionary change in morphology? This question can be addressed in species in which derived characters are present but not fixed. In rhabditid nematodes, the arrangement of the nine bilateral pairs of peripheral sense organs (rays) in tails of males is often the most highly divergent character between species. The development of ray pattern involves inputs from hometic gene expression patterns, TGFβ signalling, Wnt signalling, and other genetic pathways. In C. briggsae AF16 and HK104 strains exhibited a wide range of phenotypes including some that were more extreme than either parental strain. Transgressive segregation was significantly associated with allelic variation in the C. briggsae homolog of abdominal B, Cb-egl-5. At least two genes that affected different elements of ray pattern, ray position and ray fusion, were linked to a second gene, mip-1. Consistent with this, the segregation of ray position and ray fusion phenotypes were only partially correlated in the RILs.Recombinant inbred lines (RILs) constructed from crosses between the variant The evolution of ray pattern has involved allelic variation at multiple loci. Some of these loci impact the specification of ray identities and simultaneously affect multiple ray pattern elements. Others impact individual characters and are not constrained by covariance with other ray pattern elements. Among the genetic pathways that may be involved in ray pattern evolution is specification of anteroposterior positional information by homeotic genes. Caenorhabditis elegans and Oscheius tipulae, cells in the vulval equivalence group that do not participate in vulval development are eliminated through fusion with the multinucleated syncytial epidermis . In C. elegans, unc-22 is located on chromosome IV and is linked to several genes known to regulate homeotic gene expression patterns, e.g. lin-49 which encodes a bromodomain protein thought to be involved in chromatin remodeling . These modeling . mip-1 mC. briggsae strains were constructed in which HK104 DNA, in the region of mip-1, has been introgressed into an otherwise AF16 background. This was accomplished through a series of backcrosses that were initiated by mating HK104 males to mip-1 (AF16) hermaphrodites. For each introgressed strain, F1 through F6 males were crossed to mip-1 (AF16) hermaphrodites. Wild-type F7 hermaphrodites were selected and propagated by self-fertilization. From each F7 hermaphrodite, multiple wild-type F8 hermaphrodites were picked. Homozygous strains were established from F8 hermaphrodites that segregated only wild-type progeny. From each set of backcrosses, a single introgressed strain was retained. These introgressed strains were scored for the position of ray 3 and for ray 3–4 fusions . Moreover, evidence for a gene affecting only ray fusion was obtained from comparisons of ray patterns exhibited by the mip-1 introgressed strains. Hence, variation in ray pattern evolution is not wholey constrained by the specification of ray identities and other ray pattern elements, such as neurotransmitter usage, also may vary independently. A similar pattern of partial constraint and flexibility has been observed for variation in eyespots size in the butterfly Bicyclus anynana [Ray pattern in  fusions . Derivedentities ,22-25,30 anynana .C. briggsae have been described [C. briggsae-like ray pattern also has been reported for C. clavopapillata [C. clavopapillata has not been observed since its first description and some authors have considered it and C. briggsae to be synonymous [C. clavopapillata is distinct from C. briggsae, the posterior position of ray 3 and its frequent fusion with ray 4 may be ancestral for these two species. This would make the C. elegans-like ray pattern in haplotype 2 an atavistic character. It should be possible to discriminate between these two models either through the characterization of additional C. briggsae strains or through the redescription and molecular characterization of C. clavopapillata.Two haplotypes of escribed  and variescribed . Strainsapillata , anothernonymous . If C. cC. briggsae. The relatively slight difference between the AF16 and HK104 phenotypes hid a wealth of genetic variation. This was evident in the nearly continuous variation exhibited in RILs for both the position of ray 3 and its fusion with ray 4. This variation included many RILs with phenotypes more extreme than either parental strain. Such transgressive segregation is common in both plants and animals and is thought to arise through fluctuating selection and/or through stabilizing selection at minor QTL when directional selection at major QTL overshoots a phenotypic optimum [C. elegans, ray 3 precursor cells are born at the junction of the mab-5 and egl-5 expression domains [C. elegans ray pattern to phenocopy that of C. briggsae [Cb-egl-5 and variation in ray pattern. As Cb-egl-5 and Cb-mab-5 are closely linked, variation in either or both of these genes may be responsible for the observed association. Alternatively, linkage to Cb-egl-5 may be coincidental, and ray pattern variation in C. briggsae may not result from variation in homeotic gene expression patterns. The best test of these alternative models will be the high resolution mapping of the allelic variants responsible for ray pattern variation.Regardless of their evolutionary history, haplotypes 1 and 2 provided an entrée to the genetics of morphological variation of ray pattern in  optimum . The tra domains ,47. Thes domains  and somebriggsae ,26. We hmip-1 marker assisted introgression studies. The C. elegans homolog of mip-1, unc-22, is not linked to the the homeotic gene cluster and linkage of mip-1 to Cb-egl-5 and Cb-mab-5 is unlikely. There appear to be at least two loci that affect ray pattern linked to mip-1. One of these affected both the placement of ray 3 and its fusion with ray 4. The other affected only ray fusion. These associations were not transgressive, i.e. the allelic variants linked to mip-1 antagonized the allelic variants linked to Cb-egl-5. If the transgressive segregation linked to Cb-egl-5 does result from allelic variation in one or more homeotic genes, then the antagonistic variation linked to mip-1 may be in genes that regulate homeotic gene expression patterns. A direct test of this hypothesis is possible. Several genes required for proper regulation of homeotic gene expression patterns have been identified in C. elegans [C. briggsae homologs of these genes could be tested for association with ray pattern variation in the RILs. Ideally, these tests would be integrated into a genome wide screen for variant loci with effects on ray pattern. This will require the enhancement of genetic resources available for C. briggsae.Loci not linked to the homeotic gene cluster also must be involved in ray pattern variation. Direct evidence for this was obtained from the  elegans ,37,38,49Caenorhabditis provides a powerful model for the study of morphological evolution. Macroevolutionary comparisons between species and microevolutionary analyses of variation within species are possible. Augmenting these approaches is a detailed understanding of the genetic and cellular basis of ray pattern development in C. elegans. In C. briggsae, intraspecific variants have been characterized that affect the expression of ancestral and derived ray patterns. These variants have a complex genetic basis involving multiple genes. Some of these genes exhibit transgenic segregation, some affect all of elements of ray pattern, and some that affect only a subset of ray pattern elements. At least one gene that affects ray pattern variation in C. briggsae is linked to the homeotic gene cluster. Thus, ray pattern variation may result from altered expression patterns of homeotic genes. Further characterizations of the genetics of ray pattern variation will test this model and will address interactions between different genes that impact ray pattern in C. briggsae.Ray pattern in C. briggsae strains AF16, and HK104 are available from the Caenorhabditis Genetics Center [E. coli strain OP50. Recombinant inbred lines (RIL) were constructed starting with HK104 males mated to sperm-depleted AF16 hermaphrodites [s Center . These shrodites . Each RIRay pattern phenotypes were scored using differential interference contrast optics at a magnification of 400× . Right aCb-egl-5, CAGGGAGCGGACAACTTCAAAGG and GGACACAGCCCAGGATTAGCGAC, respectively, were designed based on genome sequence data from C. briggsae strain AF16 [Forward and reverse primers for PCR amplifications ain AF16 . AmplifiCb-egl-5 allelic variation with ray pattern variation were determined online at [mip-1 introgressed strains were compared using reciprocal chi-squared tests using Excel v10.1.0 .Pearson's product moment correlation cofficients between ray pattern elements in RILs and Wilcoxon nonparametric tests of the association between nline at . Frequennline at . Ray patdll = distalless; en = engrailed; sal = spaltRIL = recombinant inbred line; QTL = quantitative trait loci; SEB: Planned and supervised all research activities. Participated in construction and genotyping of RILs. Reviewed all statistical analyses. Wrote manuscript.CRD: Constructed, genotyped, and characterized ray pattern phenotypes of RILs. Statistical analyses of the segregation of ray pattern phenotypes and association of Cb-egl-5 variation with ray pattern variation. Reviewed manuscript.JCB: Constructed and characterized phenotypes of mip-1-assisted introgressed lines. Statistical analyses of ray pattern phenotypes of mip-1 introgressed lines. Reviewed manuscript.Cb-egl-5 genotypes and ray pattern phenotypes of RIL derived from AF16 × HK104.AF16 × HK104 RIL ray pattern data. Click here for file


Recent magnetic resonance spectroscopy (MRS) studies report that glutamine is altered in the brains of schizophrenic patients. There were also conflicting findings on glutamate in cerebrospinal fluid (CSF) of schizophrenic patients, and absent for glutamine. This study aims to clarify the question of glutamine and glutamate in CSF of first episode and drug naive schizophrenic patients.Levels of glutamine and glutamate in CSF of 25 first episode and drug-naive male schizophrenic patients and 17 age-matched male healthy controls were measured by a high performance liquid chromatography.The ratio (126.1 (median), 117.7 ± 27.4 (mean ± S.D.)) of glutamine to glutamate in the CSF of patients was significantly  higher than that (81.01 (median), 89.1 ± 22.5 (mean ± S.D.)) of normal controls although each level of glutamine and glutamate in patients was not different from that of normal controls.Our data suggests that a disfunction in glutamate-glutamine cycle in the brain may play a role in the pathophysiology of schizophrenia. Multiple lines of evidence suggest that a dysfunction in glutamatergic neurotransmission might be involved in the pathophysiology of schizophrenia -6. The ain vivo proton magnetic resonance spectroscopy (MRS), a significant increase in glutamine level was detected in the medial prefrontal cortex of never-treated schizophrenic patients compared with controls [By means of controls . In addicontrols . In contcontrols . Thus, iAlthough Kim et al.  first reIn this study, we investigated whether levels of glutamate and glutamine or ratio of glutamine to glutamate in CSF of first episode and drug naive schizophrenic patients are different from those of age-matched healthy normal controls.Twenty-five male patients with schizophrenia  and 17 age-matched male healthy subjects  with no psychiatric disease were enrolled in Uppsala University and Linkoping University Hospital, Sweden. All patients diagnosed according to the DSM-III-R were first episode and drug naive, i.e. they had never been treated with antipsychotic drugs. In the morning (8:00–9:00) from May 1997 to January 1998, CSF of subjects was obtained by lumbar puncture (L4-L5), and twelve to eighteen μL of CSF was collected with a 0.9 mm needle and the samples were immediately frozen at -80°C, as reported previously [3CN. The reaction mixture was then heated at 60°C for 2 min, and immediately supplemented with 100 μL of H2O/CH3CN (90/10) containing 0.1 % trifluoroacetic acid (TFA) to stop the reaction. Ten μL of the resultant solution was injected into the HPLC system. A reversed-phase ODS column  was used for the separation and quantification of glutamate and glutamine, and the gradient elution of the mobile phase was kept at a constant flow rate of 0.8 mL/min. Mobile phase 1a consisted of H2O/CH3CN (90/10) containing 0.1 % TFA, and phases 1b and 1c, of H2O/CH3CN (10/90) containing 0.1 % TFA and CH3CN, respectively. The time program for gradient elution was programmed as follows: 0–50.5 min 1a: 1b : 1c = 95 : 5 : 0, 50.5–55.5 min 1a : 1b : 1c = 0 : 100 : 0, and 55.5–57 min, 1a : 1b : 1c = 0 : 0 : 100. The column temperature of all columns was maintained at 35°C. Fluorescence detection was made at 530 nm with an excitation wavelength at 470 nm.Measurement of glutamate and glutamine levels were carried out according to established methods  with a sDifferences between two groups were analyzed using the Mann-Whitney U-test. The relationship between two variables was examined using Spearman correlation coefficients. A p < 0.05 was considered significant.The CSF levels (421.7 μM (median), 468.1 ± 146.1 μM (mean ± S.D.), 254.0–775.1 (range)) of glutamine in the patients were not different  from those (410.5 μM (median), 405.6 ± 108.6 μM (mean ± S.D.), 219.8–689.0 (range)) of normal controls. The CSF levels (4.17 μM (median), 4.25 ± 1.77 μM (mean ± S.D.), 2.22–8.88 (range)) of glutamate in the patients were not different  from those (5.26 μM (median), 4.73 ± 1.29 μM (mean ± S.D.), 2.54–6.51 (range)) of normal controls. However, the ratio (126.1 (median), 117.7 ± 27.4 (mean ± S.D.), 42.0–161.6 (range)) of glutamine to glutamate in the CSF of patients was significantly  higher than that (81.01 (median), 89.1 ± 22.5 (mean ± S.D.), 59.7–134.0 (range)) of controls (Table 1). Furthermore, we found significant correlations between glutamate and glutamine in normal controls  or patients .In this study, we found that the ratio of glutamine to glutamate in the CSF of first episode and drug naive schizophrenic patients was significantly higher than that of normal controls although each level of glutamine and glutamate in the CSF of patients was not significantly different from that of normal controls. To our knowledge, this is a first report demonstrating that the ratios of glutamine to glutamate in the first episode and drug naive patients are significantly higher than those of normal controls. In contrast, it was supposed earlier that alterations in CSF levels of glutamate are not so prominent compared with those in the brain . TherefoIn general, glutamine is synthesized in astrocytes from glutamate by the enzyme glutamine synthetase, found exclusively in brain glia cells. Glutamine then crosses the astrocytes to be transported into nerve cell terminals, where it is converted again into the neurotransmitter glutamate by glutaminase. It is reported that activities of glutaminase and glutamic acid decarboxylase  are significantly greater in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia than in the control group, whereas activities of glutamate dehydrogenase, glutamine synthetase, and GABA transaminase in the DLPFC of schizophrenia are not different from the control group . These fAs described in introduction, sodium-dependent amino acids transporters, located in the abluminal membranes of the blood brain barrier, are capable of actively removing amino acids from the brain ,20,21. SOur findings suggest that a dysfunction in glutamate-glutamine cycle between neuron and glia may play a role in the pathophysiology of schizophrenia, supporting the glutamate hypothesis of schizophrenia.The author(s) declare that they have no competing interests.KH conceived of the study, its design and coordination, and edited the manuscript. GE participated in the design of the study. CN and LHL recruited subjects and collected CSF samples. ES and MI assisted HPLC analysis and data analyses. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Gliomas are "intraparenchymally metastatic" tumors, invading the brain in a non-destructive way that suggests cooperation between glioma cells and their environment. Recent studies using an engineered rodent C6 tumor cell line have pointed to mechanisms of invasion that involved gap junctional communication (GJC), with connexin 43 as a substrate. We explored whether this concept may have clinical relevance by analyzing the participation of GJC in human glioblastoma invasion.Three complementary in vitro assays were used: (i) seeding on collagen IV, to analyze homocellular interactions between tumor cells (ii) co-cultures with astrocytes, to study glioblastoma/astrocytes relationships and (iii) implantation into organotypic brain slice cultures, that mimic the three-dimensional parenchymal environment. Carbenoxolone, a potent blocker of GJC, inhibited cell migration in the two latter models. It paradoxically increased it in the first one. These results showed that homocellular interaction between tumor cells supports intercellular adhesion, whereas heterocellular glioblastoma/astrocytes interactions through functional GJC conversely support tumor cell migration. As demonstrated for the rodent cell line, connexin 43 may be responsible for this heterocellular functional coupling. Its levels of expression, high in astrocytes, correlated positively with invasiveness in biopsied tumors.our results underscore the potential clinical relevance of the concept put forward by other authors based on experiments with a rodent cell line, that glioblastoma cells use astrocytes as a substrate for their migration by subverting communication through connexin 43-dependent gap junctions. Glioblastoma, the most aggressive primary brain tumor, is invariably associated with profuse and long-distance invasion of the brain parenchyma. Accordingly, this tumor has been defined as "intraparenchymally metastatic" . Intra-pWe have undertaken to identify the mechanisms of this particular type of glioma invasion, on the basis of the hypothesis that cells in the brain parenchyma may play a supportive role in this process. One key for the elaboration of this working hypothesis has been the demonstration that this intra-parenchymal invasion occurs in a non-destructive way , as this suggests that invading human glioblastoma cells may establish relationships of cooperation with their environment and in particular with resident brain cells. We have focused our attention on gap junctions because of two complementary reasons. First, evidence from peripheral tumors such as melanoma has implicated gap junctions in tumor cell migration , and linex vivo, our analysis has taken advantage of ISIS, the "intra-slice implantation system" that we have recently developed   and heterocellular coupling with astrocytes can occur. GL15 cells displayed a significantly higher invasive potential than 8-MG cells . These three tumors were precisely the ones in the series of 7 tumors to not exhibit any response to CBX treatment  were studied in brain slice culture, with or without addition of CBX or GZA. Overall, CBX treatment reduced migration indices of these four tumors by 30%  expressed abundantly both Cx43 isoforms Cx43-NP and Cx43-P1, whereas the three others  which were not invasive and did not exhibit any response to CBX treatment, expressed lower levels of Cx43-P1 isoform and did not present detectable levels of Cx43-NP. Fig. . No exprThe amount of tissue available for experimental analysis did not allow us to carry out western blotting on freshly biopsied tumor fragments. Thus Cx43 expression in the human tumor biopsies was evaluated by immunohistochemistry on paraffin embedded tissue section.Cx43 immunostaining was seen in all 4 specimens. Staining was patchy, and both the intensity of staining and the number of immunostained cells in patches differed from one sample to another. In the typical case, a positive microscopic field contained several strongly stained cells, and several unequivocally labeled cells of lower intensity Fig. . HomogenWe have next evaluated homocellular coupling between glioma cells by the scrape loading method, in which homocellular coupling is demonstrated by the diffusion of the dye from mechanically lesioned cells to their neighbors in a monolayer culture. Assay revealed extensive coupling of GL15 (homocellular coupling index: 63.75 ± 4.4 %), comparable to that displayed by astrocytes in parallel control experiments (60 ± 5.2%), whereas 8-MG cells were only weakly coupled , a result in keeping with their low level of Cx43 expression. Homocellular coupling of all cell types was strongly affected by addition of the gap junctional blocker CBX: down to 7.4 ± 1.3 % for astrocytes (p < 0.001), to 1.97 ± 0.47 for GL15 (p < 0.001) and to 2.2 ± 0.47 for 8-MG cells (p < 0.001)  / S0 = 55 ± 4.1 and 25.4 ± 5.25 for GL15 and 8 MG, respectively, p < 0.001 vs. untreated in both cases]  . C6 tumoThe main result of this study is the demonstration that functional GJC between tumor cells and host astrocytes is instrumental in the invasion of the brain parenchyma by human glioblastoma cells. Establishment of functional coupling between those two cell populations may be mediated by the gap junction protein Cx43 as ability of glioblastoma cells of various origins to migrate out of the tumor bulk into the brain parenchyma, appeared related to the level of Cx43 expression. In support of this hypothesis, overexpression of a fusion Cx43-GFP gene construct triggered a migratory behavior in otherwise non-infiltrative C6 glioma cells implanted into the brain. The similarities between those results and mechanisms of neural precursor cell migration during embryonic development suggest that glioma cells may subvert and take advantage of a physiological mechanism of cell migration into the brain. In contrast, homocellular interaction between glioma cells through gap junctions hampers their dissemination out of the tumor mass, pointing to an additional role for gap junctions, in cell-to-cell adhesion.Over the past decade, studies on GJC in gliomas have mainly addressed its role in uncontrolled cell proliferation -20, by aSeveral lines of evidence drawn from our results converge to indicate that this concept is clinically relevant, as GJC with astrocytes also plays a role in the migration of brain tumor cells for human glioblastomas. Experimental results obtained here with native human glioblastoma cells of various origins, can indeed be interpreted as a demonstration of a similar role for GJC. First, pharmacological blockade of GJC by the specific inhibitor CBX  had negaThe pharmacological approach used here had both advantages and drawbacks, which should be indicated as they may limit the conclusions to be drawn. The main advantage of that technique is the fact that it allowed us to study GJC inhibition in fresh tumor samples, in which biological properties and diversity of glioblastoma cells are well preserved, in contrast to the clonal selection and genetic variation that are commonly observed with cells lines. Conversely, we probably introduced some biological variability in the experimental system. This may be relevant to the fact that GJC inhibition with CBX did not abolish but rather reduced the rate of human glioma cell migration in our study, whereas Lin et al. 2002) obtained an "all-or-nothing" effect following genetic engineering of the C6 line. The partial effect on migration observed here may be due to the partial pharmacological inhibition consistent with the results of other studies ,25 that 02 obtainLevels of Cx43 expression were positively related to glioma invasiveness. Analysis of Cx43 expression by western blot allowed detection of two distinct isoforms of the Cx43 protein, Cx43-P and Cx43-NP. Both isoforms were detected in the four highly invasive tumors , whereas the three minimally invasive tumors  displayed no detectable levels of Cx43-P isoform. The presence of both Cx43 isoforms strongly correlated with the invasive capacities of the tumor. However, whether the migratory capacity may depend on the overall levels of expression, and/or upon the concomitant presence of both Cx43 isoforms is, at this stage, a matter of speculation. Published data on the association of either isoform with the functional state of gap junctions are conflicting. Phosphorylation of Cx43 either on serine or tyrosine residues has been reported to disrupt GJC . ThiIn the biopsy specimens, levels of Cx43 expression were different from one tumor to another, and from one zone to another within a specimen, in agreement with results reported by other authors -13. InteISIS protocol used here allowed us to gain a comprehensive three-dimensional view of the astroglial meshwork, and to identify the radial orientation of astrocytic processes toward the body of the tumors. This organization is reminiscent of the scaffolding of radial glial cells observed in the developing neocortex in vivo [in vitro [A peculiar morphological finding in this study was the phenotypic alteration of astroglial processes around tumors. Changes in the astroglial phenotype induced by glioma cells through GJC have been observed by other authors . The ISI in vivo and in viin vitro . Radial in vitro communicain vitro -40. Althin vitro ,39,41, gi.e. on astrocyte monolayer or in brain slices. Quite the contrary, heterocellular glioma-astrocytes GJC seemed to be necessary for the invasive process to occur normally, as CBX significantly blocked it in those conditions.The three different migration assays allowed us to explore the importance of intercellular interactions between migrating cells and cellular environment occurring during invasion. We have shown that inhibition of homocellular GJC in GL15 spheroids seeded on collagen IV (i.e. in the absence of astrocytes), boosted glioma cell motility, resulting in a higher centrifugal dispersion of cells. This result is in keeping with that obtained by Lin et al (2002) in an experiment in which Cx43 was shown to mediate glioma cell adherence and aggregation. However, accelerated motility of GL15 glioma cells when CBX blocked homocellular glioma/glioma GJC was not observed when glioma cells additionally formed heterocellular GJC with astrocytes, ex vivo on different supports of migration point to a functional difference between homocellular (glioma-glioma) and heterocellular (glioma-astrocytes) GJC. This may eventually be of prime therapeutic interest, by revealing mechanisms by which glioma cells disengage themselves from their neighbors in the tumor bulk, and establish new contacts with host astrocytes in order to migrate away [The purpose of our study was to determine whether the concept that functional gap junctional communication with host astrocytes facilitates invasion of malignant glioma cells put forward by other authors on the basis of experiments using a rodent glioma model , indeed apply to human glioblastoma and may, as a consequence, bear clinical relevance. This has indeed been clearly confirmed by the inhibitory effect of the specific GJC blocker, carbenoxolone, on the migration of human glioblastoma cells. Results obtained ate away .in vitro using three different types of preparations, namely glioblastoma cell lines, xenografts in nude mice, and freshly biopsied tumors. Because of selection for genotypes, the original cell heterogeneity of gliomas is restricted in xenografts and absent from cell lines. These models, therefore, do not fully reproduce the cell content of in vivo gliomas but, reciprocally, they present the advantage of being homogenous, easy to handle, and to allow analysis of large numbers of cells. ISIS uniquely permits to study, in addition, the invasiveness of freshly biopsied tumors with preserved cell repertoire. In those conditions, however, investigation is limited by the amount of available tissue.The role of GJC in human glioma invasion was investigated 5 cells were seeded in 1.5% agar-coated flasks  for 7–10 days to obtain GL15 and 8-MG spheroids. Spheroids with a diameter ranging from 50–100 μm were selected for migration assays.The two human glioblastoma (GB) cell lines used in the study, GL15  and 8-MGNu/Nu mice , kindly provided to us by Dr M-F. Poupon and P. Leuraud [Seven grade III-IV human glioma biopsies xenografted and maintained subcutaneously in Swiss  Leuraud , were usFinally, four fresh human biopsy specimens were obtained from stereotactic or resection biopsies in the operating room. All samples were retrieved by the neurosurgeon from areas of enhanced contrast on magnetic resonance imaging, which correspond to a hypercellular zone on histological examination of the tumor. After retrieval, samples were immediately transferred in glioma-cell medium and prepared for implantation into brain slices. Pathological analysis of the same tumors was carried out in parallel on adjacent specimens; it systematically included immunohistochemistry for the connexin protein Cx43 in addition to usual stains. According to the WHO 2000 classification, these tumors were glioblastomas in three cases, an anaplastic grade III astrocytoma in one. For the sake of simplicity the generic term of glioblastoma will be used in the rest of the text.2, 1 mM MgCl2 10 mM glucose and 10 mM HEPES pH 7.3). Cells were then washed in a Ca2+-free HEPES solution to prevent uncoupling. After scraping with the razor blade cells were incubated in the same solution containing 1 mg/ml of Lucifer Yellow (LY)  and 1 mg/mL of Rhodamin Dextran 10,000 MV (Molecular Probes) for 1 minute. Dye transfer through GJC was assessed 10 minutes after scraping, using an Axioplan-2 microscope and appropriate filters . Fluorescent and bright light images digitized with a CoolSNAP camera  from every microscopic field were merged, and the homocellular coupling index was calculated as the ratio of the number of LY-labeled cells to the total number of cells.Functional assessment of gap junctional communication (GJC) was performed on glioblastoma cell lines. Homocellular GJC was determined by the scrape-loading/dye transfer technique ,46. BrieHeterocellular GJC was evaluated in glioma/astroglial cells co-cultures by a method adapted from Goldberg et al. (1995) . Tumor cEffects of inhibition of GJC were evaluated by treating the cultures with carbenoxolone (CBX); controls received its inactive analog, glycyrrhyzic acid (GZA)  . Migration out of tumor fragments issued from xenografted gliomas or from freshly biopsied specimens were only studied using the ISIS method.Migration of glioblastoma cell lines was analyzed in three different experimental Migration of GL15 and 8 MG cells out of spheroids deposited onto collagen-coated  cell culture dishes was analyzed over 4 days.For astrocytic co-cultures, primary astrocyte cultures were obtained from cerebral hemispheres of neonatal C57/bl6 mice  as previously described . Cells wISIS migration assay was carried out as described previously [2. The medium was changed three times a week. 24 hours after brain slice preparation, GL15 and 8-MG spheroids or small fragments of xenografted tumors and freshly biopsied specimens were deposited onto the slice surface and gently pushed with the tip of a needle, until the tumor tissue was well enveloped by the parenchyma of the slice. Cultures were maintained for 5 or 7 days for cell lines and tumor fragments, respectively.The eviously , using bIn all experimental conditions, effects of the blockade of GJC were assessed using CBX , with two negative controls: untreated and GZA supplemented to the medium instead of CBX. These compounds were added to the culture medium 24 h after glioma spheroid or tumor implantation.4 cells in 1 μL) (n = 5). Mice were sacrificed at day 8 and perfused with paraformaldehyde (4%) 8 days after implantation. Brains were sectioned serially on cryostat at 30 μm thickness as previously described [For a control experiment, we transfected the C6 glioma cell line with a plasmid vector encoding either a green fluorescent protein (GFP) fused to the carboxyl terminus of Cx43  kindly pescribed .Immunoblot analysis for Cx43 was performed on cells and tumor samples that were suspended in lysis buffer containing Tris base 50 mM, pH 7.4, NaCl 150 mM, Triton X-100 0.5%, EDTA 1 mM, protease inhibitor cocktail 0.5%  and triturated to homogeneity. Homogenates were centrifuged at 13,000 G for 10 or 30 minutes , and supernatants were aliquoted and stored at -80°C. Total protein content was determined by the Lowry method with bovine serum albumin as a standard. Total protein (30 μg) was boiled for 5 min after addition of 10% glycerol/5% mercaptoethanol and separated on SDS-PAGE 12.5% gels, transferred on PVDF membrane  under classical conditions  and immuin vivo experiments following fixation using 4% paraformaldehyde in PBS at 4°C . For fluorescent immunostaining, cultures were rinsed several times in PBS, then incubated for 1 hour in PBS containing 10% normal horse or goat serum and 0.6% Triton X-100 (Sigma). They were incubated overnight at 4°C with monoclonal antibodies against vimentin , or polyclonal antibodies against glial fibrillary acidic protein-GFAP  or Green Fluorescent Protein-GFP . After 3 washes in PBS, they were then incubated with a PBS solution containing anti-rabbit or anti-mouse immunoglobulin conjugated either to fluorescein isothiocyanate-FITC, AMCA , or to cyamidine-Cy3 . For human biopsies, immunocytochemical detection of Cx43 was carried out on formalin-fixed, paraffin-embedded 5 μm sections. Antigen-retrieval consisted in heating slides immersed in citrate buffer (pH 6) in a microwave oven (3 × 5 min at 750 W). To block non-specific binding sites slides were incubated in PBS containing 10% BSA. A primary polyclonal antibody (Zymed) directed against the intracellular portion of the Cx43 molecule was applied (1:200) to sections overnight. Bound primary antibodies were detected by a Vector Elite ABC kit  following the manufacturer's instructions and using DAB as the chromogen. Slides were lightly counterstained in Harris's hematoxylin. Negative controls had the primary antibodies omitted. A specimen of normal human cortex from epilepsy surgery showed diffuse staining of astrocytic cells accentuated at perivascular and superficial locations, corresponding to astrocytic endfeet and the glia limitans, respectively.Immunohistochemistry was carried out on cell and slice cultures and brain slices from 0) / S0, where S is the maximum area containing migrating glioma cells and S0 is the initial tumoral mass area, this ratio compensating for the heterogeneous sizes of implanted spheroids and tumor fragments, and (ii) the number of invading cells (cells/mm of S0 perimeter). For collagen-coated migration assay and astrocytic co-cultures, only the maximum area of invasion was assessed. The statistical significance was evaluated using Mann & Whitney test and Student unpaired t test.Fluorescent microscopy on a Zeiss Axioplan2 microscope and image analysis using the KS.400 (3.0 version) software were performed to quantify invasion of glioma cells, according to techniques that have been detailed elsewhere . Two invROconceived and designed the study, performed acquisition, analysis and interpretation of data and drafted the manuscript. CCconceived and designed the study, performed acquisition, analysis and interpretation of data and drafted the manuscript. JSGparticipated in the design of the study and carried out xenografts in nude mice. SdB participated in the design of the study. SP provided human biopsies tissue and diagnosis. LV participated in the design of the study. MT have been involved in revising the article critically for important intellectual content. MPconceived, designed and coordinated the study, drafted the manuscript and have given final approval of the version to be published. All authors read and approved the final manuscript.


Anyone who has used a search engine quickly becomes familiar with both their power and limitations. A key-word search for “bush kerry butterfly county” turns up almost twenty-eight thousand documents, but a scant few of them are of any interest to the botanist studying the butterfly bush in County Kerry. The problem is more circumscribed but no less significant within specialized databases, such as the fourteen-plus million medical journal articles catalogued by PubMed.It is not so much that the literature is too vast, but that the search strategies are too weak. A simple list of key words used to tag and retrieve a document cannot begin to capture the richness of the information within, especially when that wealth is expressed in syntactically complex sentences like “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”But there is another way. Rather than simply extracting a limited list of key words from an article's abstract, the entire text of the document can be categorized into classes: some words represent entities  and others relationships . The entire set of entities and relationships can be linked to create a map of the information within the document, which, like a physical map, captures some of the complexity of the territory it describes.Caenorhabditis elegans.Humans excel at this type of concept mapping, but their labors are slow and expensive. In this issue, Paul Sternberg and colleagues at the California Institute of Technology  describe a computer-based system that performs the same task, and show that it is almost as good as humans at mapping out the scientific literature concerning the laboratory nematode, C. elegans, representing 60% of the entire literature.Sternberg's system, called Textpresso, includes 33 categories of terms, both of entities and relationships, and a full list of all possible examples for each entity  and relationship . This collection, called an ontology, is then applied to sentences within the text of a document to map out the relationships within—for instance, the mention of two genes within a sentence along with any form of the word stem “regulat-” indicates that one gene probably regulates the other, and the sentence is marked accordingly. With scores of tags applied, the full markup of a sentence is typically much longer than the sentence itself. Currently, Textpresso has marked up almost 4,000 full-text articles on daf-16, one enters the terms “daf-16” and “association.” Textpresso returns 125 publications, with citations and links to the articles. (Textpresso is available at www.textpresso.org.) The results can be further refined by adding other entities or relationships, as well as by specifying author, journal, or year of publication.The final result is a document that can be queried in subtle ways impossible with mere key words. For instance, to find entities  that interact with the aging-related gene C. elegans, can be used for literature analysis of other model organisms. Finally, the fully annotated literature within a field is not only a repository of scientific facts, but also a data mine of human communication, which can be queried for patterns having little to do with model organisms and much to do with how scientists communicate with each other.Textpresso's ability to find relevant documents, and ignore irrelevant ones, is still not as great as an expert human curator of the same literature. But the system can be constantly tweaked to get better and better. This process requires human intervention, and the Caltech team does not think this is likely to be automated anytime soon. On the other hand, the structure of Textpresso, and to some extent the ontological lists from