The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins.

BACKGROUND: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. RESULTS: The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. CONCLUSIONS: Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.

The nonfermentable dietary fiber hydroxypropyl methylcellulose modulates intestinal microbiota.

Diet influences host metabolism and intestinal microbiota; however, detailed understanding of this tripartite interaction is limited. To determine whether the nonfermentable fiber hydroxypropyl methylcellulose (HPMC) could alter the intestinal microbiota and whether such changes correlated with metabolic improvements, C57B/L6 mice were normalized to a high-fat diet (HFD), then either maintained on HFD (control), or switched to HFD supplemented with 10% HPMC, or a low-fat diet (LFD). Compared to control treatment, both LFD and HPMC reduced weight gain (11.8 and 5.7 g, respectively), plasma cholesterol (23.1 and 19.6%), and liver triglycerides (73.1 and 44.6%), and, as revealed by 454-pyrosequencing of the microbial 16S rRNA gene, decreased microbial α-diversity and differentially altered intestinal microbiota. Both LFD and HPMC increased intestinal Erysipelotrichaceae (7.3- and 12.4-fold) and decreased Lachnospiraceae (2.0- and 2.7-fold), while only HPMC increased Peptostreptococcaceae (3.4-fold) and decreased Ruminococcaceae (2.7-fold). Specific microorganisms were directly linked with weight change and metabolic parameters in HPMC and HFD mice, but not in LFD mice, indicating that the intestinal microbiota may play differing roles during the two dietary modulations. This work indicates that HPMC is a potential prebiotic fiber that influences intestinal microbiota and improves host metabolism.

Hypocholesterolemic effects of hydroxypropyl methylcellulose are mediated by altered gene expression in hepatic bile and cholesterol pathways of male hamsters.

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.

Quantification of the sulfated cholecystokinin CCK-8 in hamster plasma using immunoprecipitation liquid chromatography-mass spectrometry/mass spectrometry.

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 gene expression in rat kidney: influence of strain, gender, and diet.

In order to gain better insight into factors (strain, gender, and diet) influencing background variability in kidney gene expression, we examined the transcriptomes of male and female Crl:CD(SD)IGSBR (Sprague-Dawley [SD]) and CDF(Fischer 344)/CrlBR rats maintained for 19 days on three different diets (ad libitum [AL], diet restriction-75% of AL, and casein-based phytoestrogen-free diet). Kidney RNA was analyzed using Agilent Rat oligo microarrays (approximately 20,000 genes). Principal component analysis demonstrated that strain and gender have the most impact on the variability in gene expression, while diet had a lesser effect. The majority of the affected genes differed by a magnitude of four-fold or less between strains/gender, with some previously known to be sex-hormone regulated (SLC22A7 and SLC21A1). One gene of particular interest was ornithine decarboxylase, a significant marker of cell proliferation and tumor promotion, which was expressed at an 18-fold greater level in SD rats. Further analysis revealed that the difference in expression was due to the use of an alternate polyadenylation signal resulting in the production of two different sizes of transcripts. These results demonstrate that gender and strain have significant influence on gene expression which could be a confounder when comparing results, especially when it involves predictive fingerprint/patterns.

Effects of cationic hydroxyethyl cellulose on glucose metabolism and obesity in a diet-induced obesity mouse model.

BACKGROUND: To investigate the effect of a new soluble fiber, namely cationic hydroxyethyl cellulose (cHEC), on weight loss and metabolic disorders associated with obesity using a high-fat diet-induced obese mouse model. METHODS: Obese male C57BL/6J (B6) mice were fed high-fat (60% kcal) diets supplemented with cHEC for 5 weeks. Body weight, energy intake, mesenteric adipose and liver weights, plasma cholesterol, plasma insulin, glucose, adiponectin, and leptin were assessed to determine the effects of cHEC. Hepatic and fecal lipids were also analyzed to investigate the effect of cHEC on lipid absorption and metabolism. RESULTS: Supplementation of the high-fat diet with cHEC resulted in significant weight loss in obese mice. In addition, significant decreases were seen in mesenteric adipose and liver weights, as well as concentrations of plasma cholesterol and hepatic lipids. A significant improvement in glucose homeostasis, insulin sensitivity, and leptin concentrations were observed at 4% cHEC. Moreover, increases in fecal excretion of total bile acids, sterols, and fats indicated altered fat absorption when cHEC was supplemented in the diet. CONCLUSIONS: We have shown in the present study that cHEC reduces body weight, improves insulin sensitivity, and prevents the development of metabolic syndrome. Furthermore, the effects of cHEC on glucose and lipid homeostasis in B6 mice are mediated by improvements in leptin sensitivity resulting from reduced fat absorption.

Effects of cationic hydroxyethyl cellulose on dyslipidemia in hamsters.

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; P < 0.05), CYP51 (lanosterol 14α-demethylase; 6-fold of control; P < 0.05), 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.

Effect of hydroxypropyl methylcellulose on obesity and glucose metabolism in a diet-induced obesity mouse model.

BACKGROUND: To investigate the effect of hydroxypropyl methylcellulose (HPMC) on weight loss and metabolic disorders associated with obesity using a high-fat diet-induced obese mouse model under a high-fat diet regimen. METHODS: Obese male C57BL/6J (B6) mice were fed either a high-fat (60% kcal), low-fat (10% kcal), or high-fat diet plus HPMC (4% and 8%) for 5 weeks. Body, mesenteric adipose, and liver weights were determined at the end of the study. In addition, plasma cholesterol, insulin, glucose, adiponectin, and leptin were analyzed to determine the effects of HPMC. Hepatic and fecal lipids were measured to determine the effect of HPMC on lipid absorption and metabolism. RESULTS: Supplementation of the high-fat diet with 4% and 8% HPMC resulted in significant weight loss in obese B6 mice. Furthermore, significant decreases were seen in adipose (30%-40%), liver weights (15%-26%), and concentrations of plasma cholesterol (13%-20%) and hepatic lipids (13%-36%). Supplementation with 8% HPMC led to significant improvements in glucose homeostasis and leptin concentrations. Reductions in plasma cholesterol, glucose, and insulin levels were strongly correlated with reduced leptin concentrations. Moreover, increases in fecal secretion of total bile acids, sterols, and fats indicated altered fat absorption when HPMC was incorporated in the diet. CONCLUSION: The data indicate that HPMC not only reduces body weight, but also normalizes the metabolic abnormalities associated with obesity and suggest that the effects of HPMC on glucose and lipid homeostasis in B6 mice are mediated by improvements in leptin sensitivity resulting from reduced fat absorption.

Dietary hydroxypropyl methylcellulose increases excretion of saturated and trans fats by hamsters fed fast food diets.

In animal studies, hydroxypropyl methylcellulose (HPMC) intake results in increased fecal fat excretion; however, the effects on dietary saturated fatty acids (SATs) and trans-fatty acids (TRANS) remain unknown. This study investigated the effect of HPMC on digestion and absorption of lipids in male Golden Syrian hamsters fed either freeze-dried ground pizza (PZ), pound cake (PC), or hamburger and fries (BF) supplemented with dietary fiber from either HPMC or microcrystalline cellulose (MCC) for 3 weeks. We observed greater excretion of SATs and TRANS by both diets supplemented with HPMC or MCC as compared to the feed. SAT, TRANS, and unsaturated fatty acids (UNSAT) contents of feces of the PZ diet supplemented with HPMC were 5-8 times higher than diets supplemented with MCC and tended to be higher in the PC- and BF-HPMC supplemented diets as well. We also observed significant increases in fecal excretion of bile acids (2.6-3-fold; P < 0.05), sterols (1.1-1.5-fold; P < 0.05), and unsaturated fatty acids (UNSAT, 1.7-4.5-fold; P < 0.05). The animal body weight gain was inversely correlated with the excretion of fecal lipid concentrations of bile acids (r = -0.56; P < 0.005), sterols (r = -0.48; P < 0.005), SAT (r = -0.69; P < 0.005), UNSAT (r = -0.67; P < 0.005), and TRANS (r = -0.62; P < 0.005). Therefore, HPMC may be facilitating fat excretion in a biased manner with preferential fecal excretion of both TRANS and SAT in hamsters fed fast food diets.

Determination of F2-isoprostanes in urine by online solid phase extraction coupled to liquid chromatography with tandem mass spectrometry.

F(2)-isoprostanes are a unique class of prostaglandin-like compounds formed in vivo, which have been established as biomarkers of oxidative stress. Accurate analysis has been challenging due to lack of specificity for the isoforms of isoprostanes and lengthy sample preparation procedures to enable trace quantitative analysis. A quantitative analytical method was developed for the determination of F(2)-isoprostanes in rat and hamster urine by online solid phase extraction (SPE) coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS). The online SPE LC-MS/MS procedure has significant advantages over alternative methods with respect to specificity, sensitivity, simplicity, and speed. The assay enables the detection of iPF(2alpha)-III, iPF(2alpha)-IV, and iPF(2alpha)-VI over a linear dynamic range of 0.1-50 ng/mL in rat urine samples. This range covers the basal levels of these F(2)-isoprostanes. The limit of quantitation (LOQ) for the standard isoprostanes was about 0.3 ng/mL. The average recoveries ranged from 73 to 115% depending upon the individual F(2)-isoprostane isomers in rat urine. Additionally, the method was used to determine increases of endogenous urine iPF(2alpha)-VI and iPF(2alpha)-III in hamsters challenged with either low-fat or high-fat diets.

Dietary fiber improves lipid homeostasis and modulates adipocytokines in hamsters.

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.

Kinetic characterization of compound I formation in the thermostable cytochrome P450 CYP119.

The kinetics of formation and breakdown of the putative active oxygenating intermediate in cytochrome P450, a ferryl-oxo-(pi) porphyrin cation radical (Compound I), have been analyzed in the reaction of a thermostable P450, CYP119, with meta-chloroperoxybenzoic acid (m-CPBA). Upon rapid mixing of m-CPBA with the ferric form of CYP119, an intermediate with spectral features characteristic of a ferryl-oxo-(pi) porphyrin cation radical was clearly observed and identified by the absorption maxima at 370, 610, and 690 nm. The rate constant for the formation of Compound I was 3.20 (+/-0.3) x 10(5) m(-1) s(-1) at pH 7.0, 4 degrees C, and this rate decreased with increasing pH. Compound I of CYP119 decomposed back to the ferric form with a first order rate constant of 29.4 +/- 3.4 s(-1), which increased with increasing pH. These findings form the first kinetic analysis of Compound I formation and decay in the reaction of m-CPBA with ferric P450.