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Three protein emulsifiers encapsulating β-carotene (BC) with accompanying lipids into nanoemulsions (NEs) or without lipids into nanoparticles (NPs) were fabricated to study the effect of the type of interfacial protein on carrier design and the structure remodeling during digestion on the overall uptake and metabolism of BC in Caco-2 cells. BC-loaded micelles and micellar-like aggregates were collected after in vitro digestion and applied to Caco-2 cell monolayers. The digestion process significantly enhanced the cellular uptake of BC by 1.2-2.2 times and 4.1-8.2 times loaded in NEs and NPs, respectively. Whey protein isolate-based carriers improved the absorption but decreased the metabolism of BC to retinyl palmitate. The presence of lipids was found to improve metabolism and aid the transport of retinoids to the basolateral side of Caco-2 monolayers. Understanding the transportation behavior of the protein-based nanocarries after digestion may contribute to the design of biosafe carriers with higher bioavailability to deliver lipophilic nutrients.

The present study investigated the potential of potato peel powders, high in bioactive phenolic compounds and glycoalkaloids, to reduce weight gain in mice consuming a high-fat diet. Potato peel powders were prepared from the following fresh commercial potato varieties by hand-peeling and then freeze-drying and grinding the peels into powder: non-organic (conventionally grown) gold, red, and Russet and organically grown Russet. Mice diets (25% fat by weight) were supplemented with either 10 or 20% potato peel powders for 3 weeks. In comparison to the control diet, the isocaloric and isonitrogenous peel-containing diets induced a reduction in weight gain that ranged from 17-45% (10% peel diets) to 46-73% (20% peel diets), suggesting that differences in weight gain are associated with the potato peel source and peel concentration of the diet. Weight reductions were accompanied by reduced epididymal white adipose tissue ranging from 22 to 80% as well as changes in the microbiota analyzed using next-generation sequencing and in obesity-associated genetic biomarkers determined by the quantitative real-time polymerase chain reaction. Safety aspects and possible mechanisms of the antiobesity effects are discussed in terms of the composition of the bioactive potato peel compounds, which were determined using high-performance liquid chromatography. The results suggest that potato peels, a major peeling byproduct of potato processing used to prepare fries, chips, and potato flour, that showed exceptionally high antiobesity properties in fat mice, have the potential to serve as an antiobesity functional food.

The potential for apple peels to mitigate the deleterious effects of a high-fat diet in mice was investigated here. Mice were fed a high-fat diet supplemented with apple powders from three apple varieties or a commercial apple polyphenol. Polyphenols were characterized using colorimetric assays and high-performance liquid chromatography. Mice were tested for standard metabolic parameters. There was a dose response to dietary apple peels, with the higher intake leading to reduced weight gain and adipose tissue mass relative to the lower intake, but none of the treatments were statistically different from the control. The gene expression of liver enzyme stearoyl-CoA desaturase (Scd-1) was correlated with adipose weight, and liver enzyme cytochrome P51 (Cyp51) was downregulated by the apple diets. The feces from a subset of mice were analyzed for polyphenols and for bacteria taxa by next-generation sequencing. The results revealed that the makeup of the fecal microbiota was related to the metabolism of dietary polyphenols.

Orange carrots are well known for their nutritional value as producers of β-carotene, a vitamin A precursor. Lesser known, is their ability to accumulate antioxidants such as chlorogenic acid. Chlorogenic acid is produced through the same biosynthetic pathway that produces lignins, anthocyanins, flavonols and isoflavonoids, the phenylpropanoid pathway. This pathway can be induced by UV-B light and wounding to produce large amounts of chlorogenic acid. We identified a number of the carrot structural genes in this pathway and their proposed regulators in carrot root slices and analyzed their expression in response to UV-B light exposure using real-time PCR. Our results indicate that not only does the expression of the structural genes correlate with their proposed regulators, including DcHY5 and others, but also correlate with chlorogenic acid production. Our results indicate that the HY5 — UVR8 signaling network may exist and function in carrot roots.

The relationship between the intestinal microbiota and the hypocholesterolemic and antiobesity effects of whole grape seed flour from white and red winemaking was evaluated. Male Golden Syrian hamsters were fed a high-fat (HF) control diet or a HF diet supplemented with 10% partially defatted grape seed flours from either Chardonnay (ChrSd) or Cabernet Sauvignon (CabSd) grapes for 3 weeks. The numbers of total bacteria and relative abundances of Bifidobacterium spp., Lactobacillus spp., and Firmicutes in feces were significantly lower, while the relative abundance of Bacteroides fragilis was greater than the control from feeding the ChrSd diet. The ratio of Firmicutes/Bacteroidetes (F/B) was lower in the ChrSd diet. There were significantly positive correlations between Lactobacillus spp., ratio of F/B, and plasma total- and LDL-cholesterol and liver weight. The reduction of Lactobacillus spp. by the ChrSd diet was accompanied by inhibition of Farnesoid X receptor (FXR) signaling in the intestine as expression of intestinal fibrablast growth factor (FGF)15, positively regulated by FXR, was decreased. Expression of CYP7A1, negatively regulated by FGF15, was up-regulated in the liver, which indicates that alteration of the intestinal microbiota may regulate bile acid and lipid metabolism. These findings suggest that beneficial health effects of Chardonnay grape seed flour on HF-induced metabolic disease relate in part to modulation of intestinal microbiota and their metabolic processes.

Brasenia schreberi (蓴菜 chún cài) is an invasive aquatic weed found in the USA, but the plant has economic value in Asia where it is cultivated for food. The young leaves of B. schreberi are coated with gelatinous water-insoluble mucilage. This mucilage is a polysaccharide composed of galactose, mannose, fucose, and other monosaccharides. Because some carbohydrate gels are hypocholesterolemic, we evaluated their cholesterol-lowering properties in male hamsters fed hypercholesterolemic diets containing 2% gel coat from B. schreberi (GEL), or 1% cholestyramine (CA), or 5% hydroxypropyl methylcellulose (HPMC), and compared them to 5% microcrystalline cellulose (control) for 3 weeks. We found that very-low-density lipoprotein-, low-density lipoprotein-, and total-cholesterol concentrations in plasma were significantly lowered by GEL, CA, and HPMC compared to control. High-density lipoprotein-cholesterol concentration was lowered by CA and HPMC. Body weights and abdominal adipose tissue weight of GEL and control group animals were greater than those of the CA and HPMC groups. Fecal lipid excretion was greater in the CA and HPMC groups than in the control group. Expression of hepatic CYP51 and CYP7A1 mRNA was upregulated by CA, HPMC, and GEL, indicating increased hepatic cholesterol and bile acid synthesis. Expression of low-density lipoprotein receptor mRNA was upregulated by all treatments. These results suggest that modulation of hepatic expression of cholesterol and bile acid metabolism-regulated genes contributes to the cholesterol-lowering effects of GEL., Graphical abstract

The cholesterol-lowering effects of tomato pomace (TP), tomato seed oil (TSO) and defatted tomato seed (DTS) were determined in male Golden Syrian hamsters. Hamsters fed high-fat diets containing 10% TSO or 18% DTS were compared to a diet containing 10% corn oil and 10% microcrystalline cellulose (control 1), 42% TP were compared to 25% microcrystalline cellulose (control 2). TP, TSO and DTS reduced hepatic total cholesterol (TC) content. DTS also lowered plasma TC and low-density lipoprotein cholesterol (LDL-C) concentrations. Fecal excretion of lipid, bile acid and cholesterol increased in the DTS group compared to control 1. DTS-fed hamsters had higher levels of hepatic CYP7A1, CYP51, ABCB11, and ABCG5 gene expression than control, suggesting both hepatic bile acid and cholesterol synthesis increased due to increased fecal excretion of bile acid and cholesterol. The results suggest that protein, dietary fibre or phenolic compounds in DTS may be responsible for plasma cholesterol decrease.

Cationic hydroxyethyl cellulose (cHEC) was supplemented in a high-fat diet to determine if this new soluble fiber had an effect on hypercholesterolemia and dyslipidemia associated with cardiovascular disease using Golden Syrian hamster as an animal model. Supplementation of 3-5% cHEC in a high-fat diet for 4 weeks led to significant weight gain reduction in hamsters. In addition, significant decreases in adipose and liver weights, concentrations of plasma total, VLDL, and LDL cholesterol, and hepatic lipids were shown. No significant improvements in glucose and insulin levels were observed with cHEC; however, a significant increase in plasma adiponectin and a decrease in leptin were observed. As compared with controls, 8% cHEC-fed hamsters had greater levels of mRNA for CYP7A1 (cytochrome P450 7A1; 2-fold of control; P0.05), CYP51 (lanosterol 14α-demethylase; 6-fold of control; P0.05), and LDLR (LDL receptor; 1.5-fold of control) in the liver. These findings suggest the possibility of the use of cHEC for cholesterol reduction and beneficial effects on the cardiovascular risk factors.

Scope The effects of hydroxypropyl methylcellulose (HPMC), a highly viscous nonfermentable soluble dietary fiber, were evaluated on adipose tissue inflammation and insulin resistance in diet-induced obese (DIO) mice fed a high-fat (HF) diet supplemented with either HPMC or insoluble fiber. Methods and results DIO C57BL/6J mice were fed a HF diet supplemented with 6% HPMC or 6% microcrystalline cellulose (MCC). Gene expression analyses of epididymal adipose tissue by exon microarray and real-time PCR along with glucose and insulin tolerance and intestinal permeability were assessed. HPMC-fed mice exhibited significantly reduced body weight gain and adipose tissue weight as well as reduced areas under the curve for 2-h insulin and glucose responses. HPMC significantly decreased HF diet-induced intestinal permeability. Overall, HPMC enhanced insulin sensitivity and glucose metabolism and downregulated genes related to inflammation and immune response, adipogenesis, and oxidative stress markers. Pathway analysis of microarray data identified lipid metabolism, inflammatory disease, and acute phase response pathways as being differentially regulated by HPMC. Conclusion These results suggest HPMC consumption ameliorates HF diet effects on obesity-induced insulin resistance, adipose tissue inflammatory and immune responses, weight gain, as well as intestinal permeability.

Background The phenolic amine synephrine is a vascoconstrictor and bronchiectatic agent and holds promise as an aid to weight management and obesity reduction. Synephrine is structurally similar to the active ingredients of several commercial cold remedies. Some Citrus contain high concentrations of synephrine. An enzyme involved in synephrine biosynthesis, tyrosine decarboxylase (TYDC), is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that decarboxylates tyrosine to yield CO 2 and tyramine. We used PCR to screen, clone and sequence this gene from various synephrine producing and nonproducing Citrus species and varieties to determine if DNA sequence of this gene correlated with synephrine presence. Results PCR amplification and comparison of DNA sequence indicates DNA sequence differences that may cause production of truncated proteins to be produced in some nonsynephrine producing Citru s. Conclusion Synephrine production may be genetically determined in part by the gene for TYDC.

Cholecystokinin (CCK) and the different molecular forms of CCK are well established as biomarkers for satiety but accurate analysis has been limited by the multiple naturally occurring forms and extensive similarities to gastrin. Changes in levels of one form, CCK-8, a naturally occurring eight amino acid peptide of CCK, have been correlated with satiety responses. Endogenous CCK-8 has not been well characterized in Syrian Golden hamsters, an important model in the study of fat uptake and digestion. We have cloned and sequenced hamster CCK and identified and characterized endogenous CCK-8 from hamster plasma. Hamster CCK-8 is composed of eight amino acid residues which are highly conserved among other species. Following accurate identification and characterization of hamster CCK-8, we have developed a highly specific and sensitive immunoprecipitation based LC-MS/MS assay for its quantification. The present assay enables determination of active CCK-8 over a concentration range from 0.05 to 2.5 ng/mL in hamster plasma samples. This range covers both the basal and postprandial levels of CCK-8. Method performance validation samples were examined at three concentrations replicated over the course of 4 days. The assay accuracy (percent relative error, % RE) average was 11.3% with a precision (percent coefficient of variation, % CV) of 15.5% over all samples in this 4 day period. Additionally, the method was used to determine increases of endogenous plasma CCK-8 in hamsters challenged with a high-fat meal.

Background: The hypocholesterolemic and hypoglycemic effects of various natural and semisynthetic dietary fibers have been studied for their potential use in the prevention and improvement of metabolic syndrome. Of these dietary fibers, hydroxypropyl methylcellulose (HPMC) has been shown to lower plasma cholesterol and reduce weight gain. However, the underlying mechanisms are not known. In the present study, we examined associations between plasma adipocytokine levels and both lipid metabolism and insulin sensitivity after HPMC intake in golden Syrian hamsters. In addition, endogenous adiponectin from hamster plasma was purified and characterized. Methods: Hamsters were treated with HPMC (2% and 4% in a high-fat diet) or 2% or 4% microcrystalline cellulose (MCC; control diet) for 8 weeks. Plasma glucose, insulin, lipids, adiponectin, leptin, and hepatic lipid levels were assessed using standard techniques. Results: After 8 weeks of feeding, plasma total cholesterol and triglyceride levels in hamsters fed the 4% HPMC-supplemented diet were significantly lower than in hamsters fed the control diet. Moreover, a significant increase in adiponectin levels and a decrease in leptin levels were observed in hamsters fed the 4% HPMC-supplemented diet. Hamster adiponectin was found to be comprised of 244 amino acid residues with an apparent molecular weight of 30 kDa, consistent with the adiponectin reported in other species. Conclusions: Reductions in plasma cholesterol and triglyceride levels were correlated with a decrease in plasma leptin and an increase in adiponectin. These results suggest that adipocytokines are regulated by HPMC and may play a pivotal role in the hypocholesterolemic effect.

Tomato (Solanum lycopersicum, formerly Lycopersicon esculentum) cv. VFNT Cherry sepals, when cultured in vitro between 16 degrees C and 22 degrees C, change their genetic programme to that of ripening fruit. Previously regulation of a number of transcription factors and a putative G-protein-coupled receptor that may be involved in tomato fruit ripening and cool-temperature sepal morphogenesis had been revealed. Many of those genes such as TAG1, TM4, TM6, AP2-like (LeAP2FR), YABBY2-like (LeYAB2), and LeCOR413-PM1 have not been investigated for ethylene regulation. Ethylene-independent, regulated transcripts may be part of an early signalling process induced or de-repressed by cool temperature that causes a switch in the genetic programme of the sepals. In this paper, ethylene regulation of a number of these and other putative signalling factors are investigated during cool-temperature-induced sepal morphogenesis. 1-Methylcyclopropene was used to block ethylene-induced gene expression by interrupting the ethylene signal transduction pathway that occurs in ripening tomato fruits and presumably in ripening sepals. Transcripts of several putative transcription factors previously shown to be up-regulated during cool-temperature-induced sepal morphogenesis (TAG1, TM4, LeAP2FR) were only slightly or not induced in 1-methylcyclopropene-treated sepals, indicating either direct or indirect ethylene regulation. Two genes, VAHOX1, a homeobox domain leucine-zipper-encoding gene, and LeYAB2, a putative zinc-finger transcription factor-encoding gene, increased in treated and untreated sepals indicating regulation by cool temperatures independently of ethylene.

Many sequences of genes now are contained in databases available to the public. These databases will be the foundation and starting points of research in the future. Extracting information from these databases is useful to all forms of genetic, biochemical, and molecular biological research. We are interested in improving the nutrition of the tomato (Lycopersicon esculentum) through an understanding of the fundamentals of the molecular biological and biochemical processes that occur during tomato fruit development. The TIGR Tomato Gene Index has been of much use to our laboratory in determining new areas of molecular analysis for this research. We have examined this database for genes involved in ripening by using the nucleic acid and protein sequence searching software, BLAST, and by comparing gene expression information contained within the database. In this article, we report a number of previously unobserved genes that possibly are involved in tomato fruit development. Transcripts for MADS Box genes, zinc-finger genes, homeobox genes, and polycomb genes are among the transcripts that reveal upregulation during tomato fruit ripening. We believe that the information contained in this article will be valuable in considering approaches to analyzing the developmental processes involved in tomato fruit ripening.

To determine whether flavonoid-rich wine grape seed flour improves insulin sensitivity and glucose metabolism in obese mice, diet-induced obese (DIO) mice were fed a HF diet supplemented with either 10% partially defatted Chardonnay grape seed flours (ChrSd) or microcrystalline cellulose (MCC, control) for 4 weeks. The 2-h insulin and glucose areas under the curve were lowered by ChrSd indicating that ChrSd enhanced insulin sensitivity and improved glucose metabolism. ChrSd intake also reduced body weight gain, liver and adipose tissue weight, and plasma total and LDL-cholesterol concentration despite a significant increase in food intake. Exon microarray analysis of hepatic gene expression revealed down-regulation of genes related to gluconeogenesis, inflammation, triglyceride synthesis, and polyunsaturated fatty acid elongation, and up-regulation of genes related to fatty acid oxidation, cholesterol and bile acid synthesis. The expression of hepatic leptin receptor was up-regulated by the ChrSd diet sup...

The mechanisms for the hypocholesterolemic and antiobesity effects of grape seed flours derived from white and red winemaking processing were investigated using male Golden Syrian hamsters fed high-fat (HF) diets supplemented with 10% partially defatted grape seed flours from Chardonnay (ChrSd), Cabernet Sauvignon (CabSd), or Syrah (SyrSd) pomace as compared to a HF control diet for 3 weeks. Hamsters fed the ChrSd diet had significantly lowered plasma total-, VLDL-, and LDL-cholesterol concentrations compared to the CabSd, SyrSd, and control diets. The improved plasma cholesterol after ChrSd was correlated with the up-regulation of hepatic genes related to cholesterol (CYP51) and bile acid (CYP7A1) synthesis as well as LDL-cholesterol uptake (LDLR). A reduction of hepatic lipid content was associated with altered expression of the genes related to lipid metabolism. However, fecal total lipid content was not changed. Expression of ileal apical sodium bile acid transporter (ASBT) was not affected by ChrSd, indicating unchanged ileal bile acid reabsorption. The antiobesity effect of the ChrSd diet appears to be related to expression of adipogenesis- and inflammation-related genes in adipose tissue. These findings suggest that flavonoid-rich Chardonnay grape seed flour induced cholesterol-lowering, antiobesity, and anti-inflammatory health benefits and attenuation of hepatic steatosis via regulation of gene expression related to cholesterol, bile acid, and lipid metabolism in liver and adipose tissue.

The in vitro micellar cholesterol displacement assay has been used to identify peptides that may potentially reduce cholesterol in vivo. Two of these peptides, LPYPR and WGAPSL, derived from soybean protein (SP) that have been reported to displace cholesterol from micelles were tested by feeding them as a part of a hypercholesterolemic diet to mice for 3 weeks. Except reduction of very low-density lipoprotein cholesterol (VLDL-C) and triglyceride contents, the peptide-containing diets increased plasma cholesterol content with the increasing dose of the peptides. Mice fed diets supplemented with the peptides also had lower fecal bile acid excretion. Negative correlations between fecal bile acid excretion and plasma total cholesterol content (r = -0.876, P = 0.062) and non-HDL-C content (r = -0.831, P = 0.084) were observed. The mRNA levels of the genes for cholesterol and bile acid metabolism, CYP51, LDLR, CYP7A1, and LPL, were up-regulated in mice fed diets supplemented with peptides except the group fed the low dose of WGAPSL. The results suggested that higher plasma total cholesterol content possibly due to lower fecal steroid excretion as well as lower VLDL-C and triglyceride contents might due to the up-regulated expression levels of the genes CYP51, LDLR, and LPL.

The effect of hydroxypropyl methylcellulose (HPMC) on hepatic gene expression was analyzed by exon microarray and real-time PCR from livers of diet-induced obese (DIO) mice fed a high-fat (HF) diet supplemented with either 6% HPMC or 6% microcrystalline cellulose (MCC). HPMC-fed mice exhibited significantly reduced body weight gain (55% lower compared to MCC), liver weight (13%), plasma LDL-cholesterol concentration (45%), and HF diet-increased intestinal permeability (48%). HPMC significantly reduced areas under the curve for 2 h insulin and glucose responses, indicating enhanced insulin sensitivity and glucose metabolism. HPMC up-regulated hepatic genes related to fatty acid oxidation, cholesterol and bile acid synthesis, and cellular activation of glucocorticoid (bile acid recycling) and down-regulated genes related to oxidative stress, triglyceride synthesis, and polyunsaturated fatty acid elongation. In conclusion, HPMC consumption ameliorates the effects of a HF diet on intestinal permeability, insulin resistance, hepatic lipid accumulation, glucocorticoid-related bile acid recycling, oxidative stress, and weight gain in DIO mice.

The phytoene desaturase gene from Rhodobacter capsulatus was expressed in Escherichia coli and the resulting protein was purified. The purification steps involved were ammonium sulfate precipitation and ion exchange chromatography, leading to a homogenous protein of 57 kDa with high specific enzymatic activity. The purified enzyme was characterized with respect to substrate specificity and product formation. In addition to phytoene, the intermediates, phytofluene and zeta-carotene, were both converted to neurosporene, the end product of the reaction. Furthermore, 1,2-epoxy phytoene was a suitable substrate whereas the C30 diapophytoene was not. The Km values for phytoene and zeta-carotene were determined to be 33.3 and 16.6 microM, respectively. The desaturation reaction is dependent on the cofactor FAD. Oxidized nicotine nucleotides or ATP had no positive effect. The Km value for FAD was 4.9 microM. Inhibition of the desaturation reaction was observed with diphenylamine.

To identify differentially expressed hepatic genes contributing to the improvement of high-fat (HF) diet-induced hepatic steatosis and insulin resistance following supplementation of partially defatted flavonoid-rich Chardonnay grape seed flour (ChrSd), diet-induced obese (DIO) mice were fed HF diets containing either ChrSd or microcrystalline cellulose (MCC, control) for 5 weeks. The 2-h insulin area under the curve was significantly lowered by ChrSd, indicating that ChrSd improved insulin sensitivity. ChrSd intake also significantly reduced body weight gain, liver and adipose tissue weight, hepatic lipid content, and plasma low-density lipoprotein (LDL)-cholesterol, despite a significant increase in food intake. Exon microarray analysis of hepatic gene expression revealed down-regulation of genes related to triglyceride and ceramide synthesis, immune response, oxidative stress, and inflammation and upregulation of genes related to fatty acid oxidation, cholesterol, and bile acid synthesis. In conclusion, the effects of ChrSd supplementation in a HF diet on weight gain, insulin resistance, and progression of hepatic steatosis in DIO mice were associated with modulation of hepatic genes related to oxidative stress, inflammation, ceramide synthesis, and lipid and cholesterol metabolism.

Plant carotenoids are red, orange, and yellow lipid-soluble pigments found embedded in the membranes of chloroplasts and chromoplasts. Their color is masked by chlorophyll in photosynthetic tissues, but in late stages of plant development these pigments contribute to the bright colors of many flowers and fruits and the carrot root. Carotenoids protect photosynthetic organisms against potentially harmful photooxidative processes and are essential structural components of the photosynthetic antenna and reaction center complexes. In plants, some of these compounds are precursors of abscisic acid (ABA), a phytohormone that modulates developmental and stress processes (Koornneef, 1986). Carotenoids with provitamin A activity are essential components of the human diet, and there is considerable evidence that many carotenoids have anti-cancer activity. Carotenoids absorbed through the diet, and often metabolized into other compounds, are responsible for the color of familiar animals such as lobster, flamingo, and fish. Often unaware of the chemical nature of food colorants, humans consume large amounts of carotenoids added to foods primarily to please the eye (Klaui and Bauernfeind, 1981). As with other natural pigments, carotenoids attracted the attention of 19th-century organic chemists. BCarotene was isolated in 1817, and the pigments of autumn leaves were identified as xanthophylls in 1837 (Isler, 1971). Classical geneticists identified carotenoid mutants in maize, tomato, and Arabidopsis during the early part of this century. However, difficulties with the biochemical properties of the enzymes of the pathway and the generally poor viability of mutants affected in carotenoid biosynthesis during early stages of development conspired to delay development of the field. Genetic studies of carotenoid biosynthesis in bacteria (reviewed in Armstrong, 1994) have played a fundamental role in the cloning and characterization of plant carotenoid genes. Since 1991, when a bacterial probe was first used to clone a plant gene for an enzyme of the pathway, progress has been rapid. However, much remains to be learned about the environmental and developmental regulation of the pathway, and many classic mutants have not yet been characterized at the molecular level. Recent reviews (Armstrong, 1994; Bartley et al., 1994; Sandmann, 1994) and two books on methods (Packer, 1992a

Consumption of diets high in soluble dietary fibers has been suggested to induce biomarkers for anti-obesity and anti-inflammation, and improve insulin resistance. To investigate the effects of hyd...

CONTENTS INTRODUCTION . . .. ......... .. . .. . . . . . .. . .. ... . ..... ..... ....... .... . .. . .. .. .... . .. . . . . .. .... . .. . . . .. . . 288 GENE CLONING ... ......... ... ......... .. . . . .... . 288 Geranylgeranyl Pyrophosphate Synthase (GGPS) ... . ......... .. ........ 288 Phytoene Synthase (PSy) ......... . . ....... ...... ...... .. . ......... . . .. 291 Phyluene Desaturase (PDS) 292 Other Genes ....... . . . . . 292 GENETIC CONTROL. .... ... ....... ..... .. . ......... . ....... , ........ ., 293 Arabidopsis thaliana ... ......... ., 293 Lycopersicon esculentum ., 294 Zea mays .... ...... . .. ..... . . .. . .. ......... .... . . ...... . .. . ..... . . . . . 295 Other Species ., ., , 295 REGULATION OF EXPRESSION ...... .. . .. . . ......... .. . ......... .. . .. . ......... . ...... 295 Light ., 295 Photooxidative Stress.... . . . . . . . ..... . . . . . . ...... .. . . . . ...... . . . . . . ......... . . . . ....... . . . . ... ...... . . . ......... . .. ... 296 Development . . ....... , ,. ... ...... ......... ..... ... ......... ...... ... ... ... ... ... ... 296 FINAL COMMENTS ,. 297

The physiological effects of the hydrolysates of white rice protein (WRP), brown rice protein (BRP), and soy protein (SP) hydrolyzed by the food grade enzyme, alcalase2.4 L, were compared to the original protein source. Male Syrian Golden hamsters were fed high-fat diets containing either 20% casein (control) or 20% extracted proteins or their hydrolysates as the protein source for 3 weeks. The brown rice protein hydrolysate (BRPH) diet group reduced weight gain 76% compared with the control. Animals fed the BRPH supplemented diet also had lower final body weight, liver weight, very low density lipoprotein cholesterol (VLDL-C), and liver cholesterol, and higher fecal fat and bile acid excretion than the control. Expression levels of hepatic genes for lipid oxidation, PPARα, ACOX1, and CPT1, were highest for hamsters fed the BRPH supplemented diet. Expression of CYP7A1, the gene regulating bile acid synthesis, was higher in all test groups. Expression of CYP51, a gene coding for an enzyme involved in cholesterol synthesis, was highest in the BRPH diet group. The results suggest that BRPH includes unique peptides that reduce weight gain and hepatic cholesterol synthesis.

The enzyme phytoene synthase (Psy) catalyzes the formation of phytoene, an intermediate in the carotenoid biosynthesis pathway. Expression of a previously described gene (PSY1) is induced by fruit ripening in tomato (Lycopersicon esculentum). We describe here the cloning of a partial cDNA for PSY2, a gene related to PSY1. A plasmid containing the PSY2 coding region under control of a bacterial promoter complements a Rhodobacter capsulatus phytoene synthase mutant, indicating that this gene has the capacity to encode an active enzyme. We used reverse transcriptase-polymerase chain reaction followed by digestion with a restriction enzyme to determine that both PSY1 and PSY2 are expressed during tomato development. PSY1 transcripts predominate in seedlings and in late stages of fruit ripening, whereas PSY2 transcripts are relatively more abundant in mature leaves. Both genes are expressed under photooxidative conditions induced by treatment with the carotenoid biosynthesis inhibitor Norflurazon. We used polymerase chain reaction-based restriction fragment length polymorphisms and alien addition lines to map PSY2 to chromosome 2. We conclude that PSY2 is a second tomato gene encoding phytoene synthase.

Carotenoids are photoprotective and colorful pigments necessary for the survival of all photosynthetic organisms. They are vital for the protection of photosynthetic complexes and membranes from reactive oxygen species. Carotenoids also cause the red, yellow, and orange coloration in flowers and vegetables to attract pollinators and vectors for distribution of seeds. The desaturation of carotenoids is essential to impart these photochemical properties to the molecule by increasing the length of the chromophore. This review discusses carotenoid desaturation from a molecular biological perspective. With the cloning of the carotenoid isomerase, most but not all the genes involved are now in the hands of researchers. This review will give the reader knowledge of the overall reaction and up-to-date information concerning new developments in carotenoid desaturation. Keywords: ζζ-carotene; ζζ-carotene desaturase; carotenoid biosynthesis; carotenoid desaturation; carotenoid isomerase; CRTISO; 3,4-didehydrolycopene; HPPDase; hydroxyneurosporene desaturase; hydroxyphenylpyruvate dioxygenase; immutans; neurosporene; lycopene; PDS; phytoene; phytoene desaturase; phytoene synthase; phytofluene; plastid terminal oxidase; prolycopene; PSY; tetra-cis-lycopene; 3,4,3′,4′-tetradehydrolycopene; ZDS; Z-ISO

Hydroxypropyl methylcellulose (HPMC), a semisynthetic, nonfermentable soluble dietary fiber, is not absorbed by the body, but its presence in the intestinal lumen increases fecal fat, sterol, and bile acid excretions and decreases intestinal cholesterol absorption, all of which may indirectly affect hepatic lipid metabolism. We measured the expression of hepatic genes involved in cholesterol, bile acid, and fatty acid metabolism in hamsters fed diets containing 39% of energy as fat and 5% of weight as HPMC or microcrystalline cellulose (control) for 4 wk. HPMC-fed hamsters gained significantly less body weight than the control group but did not differ in food intake. They had significantly lower plasma triglyceride and total-, VLDL-, HDL-, and LDL-cholesterol concentrations and hepatic total lipid, total and free cholesterol and triglyceride concentrations than controls. Compared with controls, HPMC-fed hamsters had greater levels of mRNA for CYP7A1 (cytochrome P450 7A1; 8-fold of control; P < 0.05), CYP51 (lanosterol 14alpha-demethylase; 5.3-fold of control; P < 0.05), and HMG-CoAR (3-hydroxy-3-methylglutaryl CoA reductase; 1.8-fold of control; P < 0.05). The plasma total cholesterol concentrations from both the control and HPMC groups were inversely correlated with expression of hepatic CYP7A1 (r = -0.54; P < 0.05), CYP51 (r = -0.79; P < 0.005), and HMG-CoAR (r = -0.75; P < 0.005) genes. This suggests that HPMC supplementation affected both cholesterol and bile acid synthesis. Our data confirm that altered hepatic expression of lipid metabolism-related genes, possibly due to modulation of fecal bile acid excretion and intestinal cholesterol absorption, contributes to the lipid-lowering effects of HPMC.

This study analyzed plasma lipid profiles, genes related to cholesterol and bile acid metabolism, and inflammation in liver as well as adipose tissue from Syrian Golden hamsters fed high-fat diets supplemented with blueberry (BB) pomace byproducts including 8% dried whole blueberry peels (BBPWHL), 2% dried extract of peels (BBPX; 95% ethanol extract), and 6% residue from extracted peel (BBPEXT) compared to a diet containing 5% (w/w) microcrystalline cellulose (control). All BB diets significantly lowered plasma very low density lipoprotein cholesterol and total cholesterol concentrations. Interestingly, BB diets increased fecal lipid excretion. Hepatic CYP7A1 expression was up-regulated by all BB diets, and the expression of CYP51 was up-regulated by BBPX and BBPEXT diets, suggesting that both bile acid and cholesterol synthesis were increased. No significant changes in adipocyte gene expression related to inflammatory markers were observed with any BB diet. These data suggest that hepatic modulation of bile acid and cholesterol synthesis primarily contributes to the cholesterol-lowering effect of BB pomace byproducts.

In the initial stages of carotenoid biosynthesis in plants the enzyme phytoene synthase converts two molecules of geranylgeranyl diphosphate into phytoene, the first carotenoid of the pathway. We show here that a tomato (Lycopersicon esculentum) cDNA for a gene (Psy1) expressed during fruit ripening directs the in vitro synthesis of a 47-kDa protein which, upon import into isolated chloroplasts, is processed to a mature 42-kDa form. The imported protein is largely associated with membranes, but it can be easily solubilized by dilution or by treatment at high pH. A plasmid construct containing prokaryotic promoter and ribosome-binding sequences fused to the Psy1 cDNA complements the carotenoidless phenotype of a Rhodobacter capsulatus crtB mutant. We conclude that Psy1 encodes phytoene synthase and that this enzyme is a peripheral plastid membrane protein.

Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into zeta-carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins.

Leaves of tomato (Lycopersicon esculentum) plants grown in soil in which moisture was lowered from field capacity to levels approaching permanent wilting point show a 10-fold increase in abscisic acid (ABA) and a 60 to 70 percent decrease in rbcS and cab steady-state mRNA levels. As indicated by transcription run-on experiments, the effect occurs primarily at the transcriptional level. Similar water deficit had only a minor effect on ABA level and on rbcS and cab expression in leaves of sitiens, an ABA mutant of tomato. Expression of rbcL, the chloroplast gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, is not affected by water stress. Application of exogenous ABA results in decreased rbcS and cab expression in both wild-type and sitiens leaves. Analysis of the expression of individual members of the rbcS gene family indicates that under water-deficit conditions, expression derives primarily from only three of the five rbcS genes. Effects of dark adaptation and water deficit are additive for cab but not for rbcS expression. These results support the hypothesis that, at least under water-deficit conditions, ABA or a derivative thereof mediates a negative regulation of rbcS and cab transcription in tomato plants.

We have expressed in Escerichia coli the enzymes geranylgeranyl diphosphate synthase and phytoene synthase, from the soil bacterium Erwinia stewartii, and the two carotene desaturases phytoene desaturase and carotene zeta-carotene desaturase from Arabidopsis thaliana. We show that pro-lycopene (7,9,7',9'-tetra-cis)-lycopene is the main end product of the plant desaturation pathway in these cells. In addition, light is required in this system. Whereas in the dark mainly zeta-carotene, the phytoene desaturase product, accumulates, illumination leads to activation of this intermediate caused by its photoisomerization. zeta-Carotene then meets the stereospecific requirements of zeta-carotene desaturase and pro-lycopene is formed. In contrast, a strain of E. coli carrying geranylgeranyl diphosphate synthase, phytoene desaturase and the bacterial carotene desaturase CrtI, which mediates lycopene formation from phytoene, does not require light, nor is a poly-cis-lycopene species formed. The stereoselectivity of the plant-type desaturation pathway expressed in E. coli is the same as previously shown with chromoplast membranes. As the phytoene desaturase and zeta-carotene desaturase used originate from a system not capable of developing chromoplasts, this indicates that the poly-cis pathway of carotene desaturation may have a wider occurrence than initially believed.

Publisher Summary This chapter discusses methods for the inducible expression of carotenoid biosynthesis genes in Rhodobacter capsulatus and Escherichia coli along with novel procedures for the purification of phytoene desaturases. Carotenoid desaturases are membrane-bound enzymes in both bacteria and plants. The functional expression of Neurospora crassa and Glycine max (soybean) cDNAs coding for carotenoid desaturases in mutants of the photosynthetic bacterium Rhodobacter capsulatus have been achieved. Thus, it is logical to expect functional expression in R. capsulatus of carotenoid desaturase cDNAs from other species. In addition, it is possible that cDNAs coding for carotenoid enzymes other than desaturases could be expressed in R. capsulatus carotenoid mutants. Phytoene desaturases from various organisms catalyze a different number of desaturations. Rhodobacter capsulatus phytoene desaturase converts phytoene to neurosporene, a three-step desaturation reaction. Neurospora crassa Al-1 catalyzes six desaturations. Soybean Pds1 converts phytoene to ζ-carotene, a two-step desaturation reaction. Expression of al-1 in a phytoene-accumulating strain of R. capsulatus leads to the accumulations of lycopene and 3,4 dehydrolycopene. Expression of pds in this bacterial strain results in the accumulation of ζ-carotene. Lycopene, 3,4-dehydrolycopene, and ζ-carotene are not normally accumulated in R. capsulatus.

Photosynthetic bacteria are currently used as model systems for the study of genetic, biochemical and physical aspects of photosynthesis. In this article we briefly review genetic methods that we developed for use in Rhodobacter capsulatus and we present recent results on the molecular biology of carotenoid biosynthesis.

Red ripe tomatoes are the result of numerous physiological changes controlled by hormonal and developmental signals, causing maturation or differentiation of various fruit tissues simultaneously. These physiological changes affect visual, textural, flavor, and aroma characteristics, making the fruit more appealing to potential consumers for seed dispersal. Developmental regulation of tomato fruit ripening has, until recently, been lacking in rigorous investigation. We previously indicated the presence of up-regulated transcription factors in ripening tomato fruit by data mining in TIGR Tomato Gene Index. In our in-vitro system, green tomato sepals cultured at 16 to 22°C turn red and swell like ripening tomato fruit while those at 28°C remain green. Here, we have further examined regulation of putative developmental genes possibly involved in tomato fruit ripening and development. Using molecular biological methods, we have determined the relative abundance of various transcripts of genes during in vitro sepal ripening and in tomato fruit pericarp at three stages of development. A number of transcripts show similar expression in fruits to RIN and PSY1, ripening-associated genes, and others show quite different expression. Our investigation has resulted in confirmation of some of our previous database mining results and has revealed differences in gene expression that may be important for tomato cultivar variation. We present new and intriguing information on genes that should now be studied in a more focused fashion.

Phytoene synthase (Psy) and phytoene desaturase (Pds) are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. We report here the organ-specific and temporal expression of PDS and PSY in tomato plants. Light increases the carotenoid content of seedlings but has little effect on PDS and PSY expression. Expression of both genes is induced in seedlings of the phytoene-accumulating mutant ghost and in wild-type seedlings treated with the Pds inhibitor norflurazon. Roots, which contain the lowest levels of carotenoids in the plant, have also the lowest levels of PDS and PSY expression. In flowers, expression of both genes and carotenoid content are higher in petals and anthers than in sepals and carpels. During flower development, expression of both PDS and PSY increases more than 10-fold immediately before anthesis. During fruit development, PSY expression increases more than 20-fold, but PDS expression increases less than threefold. We concluded that PSY and PDS are differentially regulated by stress and developmental mechanisms that control carotenoid biosynthesis in leaves, flowers, and fruits. We also report that PDS maps to chromosome 3, and thus it does not correspond to the GHOST locus, which maps to chromosome 11.

Carotenoids are terpenoid pigments which are accumulated in the chloroplasts of leaves and in the chromoplasts of many flowers and fruits. Phytoene desaturase (Pds), the second dedicated enzyme in carotenoid biosynthesis, is encoded in tomato by a single copy gene. A 2 kb fragment from the tomato Pds gene, comprising 1.5 kb from the promoter and 0.5 kb from the 5' non-translated region, is able to drive developmentally regulated expression of the GUS reporter gene in transgenic tomato and tobacco plants. In tomato, high levels of Pds/GUS expression are found in organs and at stages of development where chromoplasts are formed: petals, anthers and ripening fruits. Tobacco petals and fruits, which do not contain chromoplasts, show instead low levels of Pds/GUS expression. Transgenic tobacco seedlings were subjected to treatment with a range of inhibitors of carotenoid and chlorophyll biosynthesis. The results indicate that, in green tissues, carotenoid and chlorophyll levels are tightly co-regulated and that a chemically induced arrest in pigment biosynthesis results in activation of the Pds promoter. The promoter is also induced in etiolated seedlings, which contain much lower carotenoid levels than light-grown seedlings. These data suggest that in green tissues Pds gene transcription may respond to end-product regulation.