Comparative Effects of Dietary Fat Types on Hepatic Enzyme Activities Related to the Synthesis and Oxidation of Fatty Acid and to Lipogenesis in Rats

The effects of different types of dietary fat on the activities of hepatic enzymes related to fatty acid synthesis {glucose-6-phosphate dehydrogenase (G6PDH) and acetyl-CoA carboxylase ACC)}, oxidation {acyl-CoA synthetase (AST), carnitine palmitoyl transferase (CPT), and peroxisomal β-oxidation (P βOX)}, and lipogenesis {phosphatidate phosphohydrolase (PAP), diacylglycerol acyltransferase (DGAT), and phosphocholine diacylglycerol transferase (PCDGT)}, and plasma and liver lipid levels were investigated in male Wistar rats. The animals were 6 weeks old and about 120 g of body weight, and were fed on test diets containing 20% of a mixture of tripalmitin, tristearin and corn oil (SFA), olive oil (OLI), sunflower oil (SUN), linseed oil (LIS), and sardine oil (SAR) for 2 weeks. The concentrations of plasma total cholesterol (T-CHOL), high-density lipoprotein-cholesterol (HDL-CHOL), triacylglycerol (TG) and phospholipid (PL) were generally higher in the rats fed on SEA and OLI than in those given SUN, LIS and SAR. The rats fed on OLI had a higher level of liver T-CHOL than those fed on the other fats. The liver TG content was nearly higher from the intake of SFA and OLI than from SUN, LIS and SAR, although the liver PL level was not affected by the type of dietary fat. The SFA and OLI groups had the highest activities of hepatic G6PDH and ACC, and the SAR group, the lowest activities. The activities of AST and CPT, and peroxisomal P βOX in the liver were higher in the rats fed on the LIS and SAR diets than in those given the other diets. The hepatic PAP activity was higher from the intake of OLI and SUN, and tended to be higher from SFA than from LIS and SAR. The activity of liver DGAT was higher from SFA and inclined to be higher from OLI, SUN, and LIS than from SAR, while the PCDGT activity in the liver was not effected by the type of dietary fat. The concentrations of plasma and liver TG were generally positively correlated with the activities of liver enzymes related to the synthesis of fatty acids and lipids, and negatively with those involved in fatty acid oxidation. Based on these results, it is suggested that the levels of plasma and liver TG were controlled by different types of dietary fat through changes in the hepatic enzyme activities related to fatty acid synthesis, lipogenesis, and fatty acid oxidation.     

Cytotoxicity analysis of staphylococcal bi-component β-pore forming toxins using the CHO cells expressing human lymphocyte receptor CCR5

ABSTRACT

CCR5-mediated cytotoxicity of staphylococcal bi-component toxins was investigated using human CCR5-expressing CHO cells. Cytotoxicity of rim domain loop-exchange mutants between LukE and Hlg2 indicated that loop-4 of LukE is essential for cytotoxicity in combination with LukD. Interestingly, Hlg2 showed LukF-dependent CCR5-mediated cytotoxicity, suggesting that the F-components of toxins also play a role in the cell-specific cytotoxicity.     

Identification of Novel Short Peptide Inhibitors of Soluble 37/48 kDa Oligomers of Amyloid β42

Since the soluble oligomers of 42-residue amyloid β (Aβ42) might be neurotoxins at an early stage of Alzheimer’s disease (AD), inhibition of soluble Aβ42 oligomerization should be effective in the treatment of AD. We have found by phage display that a 7-residue peptide, SRPGLRR, exhibited inhibitory activity against soluble 37/48 kDa oligomers of Aβ42. In the present study, we newly prepared 3- and 4-residue random peptides libraries and performed pannings of them against soluble Aβ42 to search for important factors in the inhibition of Aβ42 oligomerization. After the fifth round, arginine-containing peptides were enriched in both libraries. SDS–PAGE and size-exclusion chromatography indicated that the inhibitory activities of 4-residue peptides against the soluble 37/48 kDa oligomers of Aβ42 were higher than those of the 3-residue peptides, and RFRK exhibited strong inhibitory activity as well as SRPGLRR. These short peptides should be useful for the suppression of soluble Aβ42 oligomer formation.     

Clinical significance of serum transforming growth factor-β1 in lung cancer

Background: Transforming growth factor-β1 (TGF-β1) plays a critical role in human cancer development. Present study aimed to explore the clinical significance of serum TGF-β1 levels in patients with lung cancer and analyze the relationship between TGF-β1 and existing tumor markers for lung cancer. Methods: Serum was collected from 118 patients with lung cancer and 40 healthy volunteers. Serum TGF-β1 levels were measured by enzyme-linked immunosorbent assay (ELISA), and the association with various clinical characteristics was analyzed. The diagnostic value of TGF-β1 was assessed alone and in combination with existing tumor markers for lung cancer. Results: Serum TGF-β1 levels were significantly higher in patients with lung cancer compared to healthy volunteers [0.6 × 10⁵ (0.4 × 10⁵, 0.9 × 10⁵) pg/ml vs 0.5 × 10⁵ (0.3 × 10⁵, 0.7 × 10⁵) pg/ml, P = 0.040]. Although there was a positive correlation between serum TGF-β1 levels and advanced stages, the significant difference was not found between early stages and advanced stages (P = 0.116). The ability of serum TGF-β1 to discriminate lung cancer at a cutoff value of 79,168 pg/ml exhibited sensitivity of 30.6% and specificity of 97.5%. Serum TGF-β1 levels were correlated to cytokeratin fragment 21-1 (CYFRA21-1; R = 0.308, P = 0.020) and neuron-specific enolase (NSE; R = 0.558, P = 0.003). The diagnostic accuracy rates for the existing lung-tumor markers, as SCC, CYFRA21-1, and NSE, were increased from 20.0%, 34.6%, and 45.9% to 48.9%, 51.7%, and 54.5%, respectively by the inclusion of serum TGF-β1 levels. Conclusion: Quantification of serum TGF-β1 levels by ELISA may provide a novel complementary tool for the clinical diagnosis of lung cancer.     

Single-step method for β-galactosidase assays in Escherichia coli using a 96-well microplate reader

Historically, the lacZ gene is one of the most universally used reporters of gene expression in molecular biology. Its activity can be quantified using an artificial substrate, o-nitrophenyl-ß-d-galactopyranoside (ONPG). However, the traditional method for measuring LacZ activity (first described by J. H. Miller in 1972) can be challenging for a large number of samples, is prone to variability, and involves hazardous compounds for lysis (e.g., chloroform, toluene).     

Production of recombinant beta-amylase of Bacillus aryabhattai

In this study, the effects of carbon source, nitrogen source, and metal ions on cell growth and Bacillus aryabhattai β-amylase production in recombinant Brevibacillus choshinensis were investigated. The optimal medium for β-amylase production, containing glucose (7.5?g·L^?1), pig bone peptone (40.0?g·L^?1), Mg²⁺ (0.05?mol·L^?1), and trace metal elements, was determined through single-factor experiments in shake flasks. When cultured in the optimized medium, the β-amylase yield reached 925.4?U mL^?1, which was 7.2-fold higher than that obtained in the initial medium. Besides, a modified feeding strategy was proposed and applied in a 3-L fermentor fed with glucose, which achieved a dry cell weight of 15.4?g L^?1. Through this cultivation approached 30?°C with 0?g·L^?1 initial glucose concentration, the maximum β-amylase activity reached 5371.8?U mL^?1, which was 41.7-fold higher than that obtained with the initial medium in shake flask.     

In vitro stability and ex vivo absorption of thymol monoglucosides in the porcine gut

Thymol α-D-glucopyranoside (TαG) and thymol β-D-glucopyranoside (TβG) are believed to have different kinetic behaviours in the porcine gut than its parent aglycon thymol. However, recently, it was shown that concentrations of both glucosides decreased rapidly in the stomach and proximal small intestine following oral supplementation to piglets as did thymol. Yet, the stability of thymol glucosides in gut contents and their absorption route remains obscure. Therefore, a series of in vitro incubations were performed, simulating the impact of pH, digestive enzymes, bacterial activity and mucosal extracts on stability of these glucosides. Their absorption mechanisms were investigated using the Ussing chamber model in the presence or the absence of inhibitors of sodium-dependent glucose linked transporter 1 and lactase phlorizin hydrolase. Both glucosides remained intact at physiological pH levels in the presence of digestive enzymes. Recoveries from TαG and TβG were below 90% when incubated with small intestinal homogenates from the distal jejunum or from all sampled sites, respectively. However, no aglycon could be detected in these samples. Bacterial inoculum of the small intestine, on the other hand, hydrolysed TβG quickly with up to 44% of free aglycon appearing. TαG proved more resistant to porcine gastro-intestinal bacterial glucosidases with only trace amounts (<1%) of free thymol at the end of the incubations. Electrophysiological measurements in Ussing chambers did not suggest active transport of the glucosides. Mucosal TαG and TβG concentrations were unchanged between start and end of the absorption measurements. Additionally, no TαG and only a very limited amount of TβG were retrieved from the serosal side. Tissue associated concentrations, although marginal (<1% of luminal concentration), were mainly as intact glucoside or as aglycon for TαG and TβG, respectively. Addition of both inhibitors significantly increased the amount of intact glucosides retrieved from the mucosal tissues as compared to controls. In conclusion, bacterial hydrolysis was identified as the most important source of TβG loss, whereas TαG seemed less prone to degradation or absorption in these in vitro and ex vivo models.     

Study on glycosylated prodrugs of toxoflavins for antibody-directed enzyme tumor therapy

Eight novel toxoflavin glycosides, which are potential prodrugs in antibody directed enzyme prodrug therapy (ADEPT), were synthesized. The structures of all toxoflavin glycosides were characterized by (13)C NMR spectroscopy, elemental analysis, and MS. Their enzymatic hydrolysis activities were tested against beta-glucosidase (EC.3.2.1.21).     

Re-characterization of three conjugated linolenic acid isomers by GC-MS and NMR

Conjugated linolenic acids (CLN) refer to a group of octadecatrienoic acids with three conjugated double bonds. Minor positional and geometrical differences among CLN isomers make their separation and identification difficult. We have used GC-MS and NMR to study three common CLN isomers namely alpha-eleostearic acid, beta-eleostearic acid and punicic acid, finding that some signals of olefinic carbon atoms in NMR spectra were mistakenly assigned in the literature. The present study was therefore undertaken to re-characterize the location of CC double bonds and assign the chemical signals of proton and carbon atoms using (1)H NMR, (13)C NMR, (1)H-(1)H two-dimensional correlation spectra ((1)H-(1)H COSY) and (13)C-(1)H two-dimensional correlation spectra ((13)C-(1)H COSY). The geometrical structure of double bonds in these three CLN isomers was identified using homonuclear decoupling technique.     

Carbanilation of cereal beta-glucans for molecular weight determination and conformational studies

Cereal beta-glucans can form aggregates in aqueous solution. The presence of aggregates in cereal beta-glucan solutions led to inaccurate determination of molecular weights and it was believed that intermolecular hydrogen bonding caused the aggregation. To eliminate aggregates, a carbanilation method for molecular weight determination of cereal beta-glucans was developed. Wheat beta-glucan samples were selected for investigation. The carbanilation method can prevent intermolecular hydrogen bonding by blocking hydroxyl groups with phenyl carbamate groups. The carbanilates of cereal beta-glucans were prepared by the reaction of cereal beta-glucans with phenylisocyanate catalyzed by DMSO and pyridine. To avoid degradation during the carbanilation reaction, relatively mild conditions were used, which led to incomplete substitution (DS: approximately 2). However, after the carbanilation reaction, the carbanilates dissolved completely in 1,4-dioxane solution without any detectable aggregates, which allowed accurate molecular weight determination. The degree of substitution (DS) of carbanilates was determined by both a nitrogen content method and an FT-IR method. The FT-IR method proved to be the more effective for DS estimation. Using this method, the converted molecular weights of cereal beta-glucans were in good agreement with the results measured in 0.5M NaOH solution, which previously was shown to be a good solvent for cereal beta-glucans. After the carbanilation reaction, conformational changes of carbanilates were studied by static and dynamic light scattering techniques. The fractal dimension (d(f)=2.27) and the structure sensitive parameters (rho >2) suggested a porous globular structure for partially carbanilated beta-glucans.