Obtaining Bixin- and Tocotrienol-Rich Extracts from Peruvian Annatto Seeds Using Supercritical CO2 Extraction: Experimental and Economic Evaluation.

Currently, Bixa orellana L. extracts are used as a color source in the food, pharmaceutical, and cosmetic industries because they are important as a potential source of antioxidant activity. The extraction is carried out by conventional methods, using alkaline solutions or organic solvents. These extraction methods do not take advantage of the lipid fraction of annatto (Bixa orellana L.) seeds, and the process is not friendly to the environment. In this work, the objective was to obtain an extract rich in nutraceuticals (bixin and tocols) of high antioxidant power from Peruvian annatto seeds as a potential source for a functional food or additive in the industry using supercritical fluid extraction (SFE). Experiments related to extraction yield, bixin, tocotrienols, tocopherols, and antioxidant activity were carried out. The SFE was performed at 40 °C, 50 °C, and 60 °C, and 100, 150, and 250 bar with 0.256 kg/h carbon dioxide as the supercritical solvent (solvent-to-feed ratio of 10.2). Supercritical extraction at 60 °C and 250 bar presented the best results in terms of global extraction yield of 1.40 ± 0.01 g/100 g d.b., extract concentration of 0.564 ± 0.005 g bixin/g extract, 307.8 mg α-tocotrienol/g extract, 39.2 mg ß-tocotrienol/g extract, 2 mg γ-tocopherol/g extract, and IC50 of 989.96 µg extract/mL. Economical evaluation showed that 60 °C, 250 bar, and 45 min presented the lowest cost of manufacturing (2 × 2000 L, COM of USD 212.39/kg extract). This extract is a potential source for functional food production.

Kinetics Crystallization and Polymorphism of Cocoa Butter throughout the Spontaneous Fermentation Process.

The spontaneous fermentation process of Criollo cocoa is studied for its importance in the development of chocolate aroma precursors. This research supports the importance of spontaneous fermentation, which was studied through the crystallization behavior and polymorphisms of cocoa butter (CB), the most abundant component of chocolate that is responsible for its quality physical properties. The k-means technique was used with the CB crystallization kinetics parameters to observe the division of the process during the first stage (day 0-3). The experimental crystallization time was 15.78 min and the second stage (day 4-7) was 17.88 min. The Avrami index (1.2-2.94) showed that the CB crystallizes in the form of a rod/needle/fiber or plate throughout the process. CB produced metastable crystals of polyforms ß1' and ß2'. Three days of fermentation are proposed to generate Criollo cocoa beans with acceptable CB crystallization times.

Evaluation of the Miscibility of Novel Cocoa Butter Equivalents by Raman Mapping and Multivariate Curve Resolution-Alternating Least Squares.

Cocoa butter (CB) is an ingredient traditionally used in the manufacturing of chocolates, but its availability is decreasing due to its scarcity and high cost. For this reason, other vegetable oils, known as cocoa butter equivalents (CBE), are used to replace CB partially or wholly. In the present work, two Peruvian vegetable oils, coconut oil (CNO) and sacha inchi oil (SIO), are proposed as novel CBEs. Confocal Raman microscopy (CRM) was used for the chemical differentiation and polymorphism of these oils with CB based on their Raman spectra. To analyze their miscibility, two types of blends were prepared: CB with CNO, and CB with SIO. Both were prepared at 5 different concentrations (5%, 15%, 25%, 35%, and 45%). Raman mapping was used to obtain the chemical maps of the blends and analyze their miscibility through distribution maps, histograms and relative standard deviation (RSD). These values were obtained with multivariate curve resolution-alternating least squares. The results show that both vegetable oils are miscible with CB at high concentrations: 45% for CNO and 35% for SIO. At low concentrations, their miscibility decreases. This shows that it is possible to consider these vegetable oils as novel CBEs in the manufacturing of chocolates.

The Kinetics of Total Phenolic Content and Monomeric Flavan-3-ols during the Roasting Process of Criollo Cocoa.

Cocoa beans are the main raw material for the manufacture of chocolate and are currently gaining great importance due to their antioxidant potential attributed to the total phenolic content (TPC) and the monomeric flavan-3-ols (epicatechin and catechin). The objective of this study was to determine the degradation kinetics parameters of TPC, epicatechin, and catechin during the roasting process of Criollo cocoa for 10, 20, 30, 40, and 50 min at 90, 110, 130, 150, 170, 190, and 200 °C. The results showed a lower degradation of TPC (10.98 ± 6.04%) and epicatechin (8.05 ± 3.01%) at 130 °C and 10 min of roasting, while a total degradation of epicatechin and a 92.29 ± 0.06% degradation of TPC was obtained at 200 °C and 50 min. Reaction rate constant (k) and activation energy (Ea) were 0.02-0.10 min-1 and 24.03 J/mol for TPC and 0.02-0.13 min-1 and 22.51 J/mol for epicatechin, respectively. Degradation kinetics of TPC and epicatechin showed first-order reactions, while the catechin showed patterns of formation and degradation.

Formation of aromatic compounds precursors during fermentation of Criollo and Forastero cocoa.

There are three main genetic varieties of cocoa (Theobroma cacao L) used in chocolate making: Forastero, Trinitario and Criollo, which are distinguished by their aroma, an attribute that determines their quality. Criollo cocoa is of the highest quality and is used in the manufacture of fine chocolates because of its fruity aroma. The aroma of Criollo cocoa is defined by volatile compounds such as pyrazines and aldehydes, which are formed during roasting of the bean, from aroma precursors (reducing sugars and free amino acids) that are generated inside the bean via enzymatic reactions during fermentation; for this reason, fermentation is the most important process in the value chain. This review discusses the production of aroma precursors of Criollo and Forastero cocoa by studying the kinetics of spontaneous fermentation and the role of starter cultures to produce aroma precursors. Fine aroma precursors produced in the pulp during the fermentation phase will migrate into the bean when it's permeability is improved and then retained during the drying phase. Diffusion of aroma precursors into the cocoa bean may be possible, this process is mathematically characterized by the coefficient of molecular diffusion D, which describe the process of mass transfer via Fick's Second Law. The current state of knowledge is analyzed based on existing research and reports some gaps in the literature, suggesting future research that will be necessary for a better understanding of cocoa fermentation.

Evaluation of multiplex polymerase chain reaction utilising multiple targets in Mycobacterium tuberculosis direct test negative but culture positive cases: a potential method for enhancing the diagnosis of tuberculosis.

PURPOSE: To evaluate multiplex Polymerase Chain Reaction (MPCR) utilising multiple targets (IS6110, Protein b [Pab] and MPB64 genes) in Mycobacterium tuberculosis Direct Test (MTD) negative but culture positive cases and comparison of MPCR with Real-Time polymerase chain reaction (RT-PCR) for diagnosis of tuberculosis. MATERIALS AND METHODS: MPCR was carried out on 28 culture positive sputum samples. Out of 28 culture positive samples, 17 were originally reported, as MTD test negative and 11 were MTD test positive, respectively. The results of MPCR were compared with RT-PCR. To check the specificity of the tests, MPCR and RT-PCR were also evaluated with 16 non-tuberculous mycobacterial (NTM) isolates. RESULTS: Out of 28 culture positive sputum samples, MPCR was positive in all 28/28 samples, whereas RT-PCR was positive in 27/28 samples and MTD test was originally tested positive in six sputum samples and on repeating MTD testing, five more sputum samples were positive and thus total number of MTD positive were 11/28 sputum samples, respectively. All the tests were negative on evaluation with all the 16 NTMs, thus giving specificity of 100% to all the tests; sensitivity of MPCR, RT-PCR and MTD tests were 100%, 96.42% and 39.28%, respectively, in these specifically selected samples. CONCLUSIONS: MPCR may be an important tool in the rapid diagnosis of tuberculosis especially in disease endemic, resource limited countries.

Cardiovascular responses during an indoor race walking competition.

AIM: This study aims to examine the cardiovascular responses during an indoor race walking competition over the distance of 3-km for female and 5-km for male athletes. METHODS: During the Italian indoor RW Championship heart rate was monitored on eleven well trained race walkers (five men and six women) and then refereed as percentages of individuals' theoretical maximum heart rate (206-0.7·age). To provide a measure of relative intensity, five HR zones were assessed. Alterations in % HRmax both for the five and three 1000-m split distances were determined. RESULTS: During the 5-km race the athletes spent 79.7% (15 min 45 s) at HR5 (i.e., 90-100% of HRmax). Specifically, % HRmax increased by 10% in the last compared to the first 1000-m sector (P=0.006, effect size = 2.47±0.83, very large), with the first 1000-m sector lower than the subsequent ones (P=0.01, effect size=2.17 to 2.47, very large). While, for the 3-km the athletes spent 86.9% (11 min 35 s) at HR5 (i.e., 90-100% of HRmax) with no differences observed in the % HRmax between the three 1000-m sectors (P>0.01). CONCLUSION: The dissemination of performance and physical attributes identified within the present study reveal that the exercise intensity of indoor race walking competitions has a high-intensity profile and will assist coaches and athletes in formulating appropriate training, competition and recovery.

Effect of ubidecarenone oral treatment on aerobic power in middle-aged trained subjects.

BACKGROUND: Coenzyme Q10 (CoQ10) plays an important role in oxidative mithocondrial phosphorylation and prevents lipid peroxidation in biological membranes. During sustained physical exercise, reactive oxygen species (ROS) production increase through several mechanism; one of them is the purine nucleotide cycle activation by shifting xanthine-dehydrogenase to xanthine-oxidase during AMP breakdown. The aim of this study was to evaluate the effect of CoQ10 treatment on aerobic power. EXPERIMENTAL DESIGN: according to a single blind study design, 28 health male cyclists were randomized into two groups (CoQ10 or placebo) and remained on treatments for eight weeks; there were 5 drop-outs and only 23 subjects were completely evaluated. Before and at the end of the eight weeks, cyclists underwent cardiopulmonary exercise testing. MEASURES: a software system performed the necessary calculations to obtain the following parameters: oxygen uptake, CO2 production, minute ventilation, oxygen ventilatory equivalent, carbon dioxide ventilatory equivalent, oxygen pulse. Finally oxygen peak and anaerobic threshold were determined. Moreover blood inosine, hypoxanthine, xanthine, lactate and CoQ10 levels were measured before and immediately after each test. RESULTS: The results of this study showed that at the end of the eight weeks there was no difference between the two groups concerning physiological and metabolic parameters, but muscular exhaustion was reached at higher workloads in the CoQ10 group. CONCLUSIONS: In our experience ubidecarenone oral treatment does not improve aerobic power. The little improvement of tolerance to higher workloads may be due to the antioxidant activity of CoQ10.

Can oxypurines plasma levels classify the type of physical exercise?

BACKGROUND: A laboratory-based model able to describe muscle energy status during physical exercise and changes in myofibrillar composition in response to training would be desirable. Lactate and ammonia concentrations are not sufficient for a comprehensive knowledge of these systems. All muscle fibres, irrespective of the type, show ATP depletion and IMP accumulation following exhausting muscular exercise with quantitative differences due to the different concentrations of deaminase. We studied the plasma concentration of metabolites of oxypurine cascade to test their reliability to classify different exercises. METHODS: We studied 52 athletes, measuring plasma metabolites at the beginning and at the end of their specific field exercise (cycle pursuers, 8 cases; soccer players, 19; marathon runners, 25). K3EDTA-blood samples were assayed for plasma hypoxanthine, xanthine, and inosine, using an HPLC technique, as well as ammonia and lactate by means of enzymatic methods. RESULTS AND CONCLUSIONS: Basal oxypurines levels were not different in relation to any specific physical exercise. Post-exercise oxypurines, namely hypoxanthine, were more precise predictors of muscle energy exhaustion than strain intensity or duration. Plasma levels of hypoxanthine may be elevated also in the presence of normal xanthine and uric acid concentrations, due to an exhaustion of the enzymatic pathway, to a reduced activity of xanthine-oxidase or finally to a substrate-dependent inhibition of the process.

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52.

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces diverse retinoic acid metabolites in multiple tissues of the Sprague-Dawley rat.

Retinoic acid metabolism was examined in microsomes prepared from four retinoid target tissues of male Sprague-Dawley rats removed 3 days after a single exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 5 or 80 micrograms/kg, ip). Microsomes from all four tissues catalyzed increased rates of retinoic acid metabolism, with the degree of induction following the order: liver > lung = kidney = testis. The responses were tissue-specific with respect to the metabolites affected, the effects of dose, and the substrate used, [3H]retinoic acid or [3H]retinoic acid bound with cellular retinoic acid-binding protein. For example, neither 4-hydroxy- nor 18-hydroxy-retinoic acid increased in testis; 4-hydroxy- but not 18-hydroxy-retinoic acid increased in liver; and both 4-hydroxy- and 18-hydroxy-retinoic acid increased in kidney and lung. This ability of TCDD to affect diverse retinoic acid metabolites in multiple tissues, including those from a physiologically relevant substrate, holocellular retinoic-acid binding protein, strengthens the possibility that one aspect of TCDD toxicity involves altering the metabolism of retinoic acid.

Enzymes and binding proteins affecting retinoic acid concentrations.

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alcohol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low Km substrate.

Identification of 9-cis,13-cis-retinoic acid as a major circulating retinoid in plasma.

In the course of measuring the concentrations of retinoic acids (RA) in bovine plasma, a major peak was observed which comigrated with 9-cis-RA on normal-phase high-performance liquid chromatography. Rechromatography of this retinoic on reverse-phase high-performance liquid chromatography showed that it was distinct from 9-cis-,13-cis-, and all-trans-RA, but comigrated with 9-cis,13-cis-retinoic acid (9,13-di-cis-RA). This retinoid was identified as 9,13-di-cis-RA based on its chemical, spectral, and chromatographic properties. Plasma concentrations of 9,13-di-cis-RA increased from < or = 0.5 ng/mL at birth to 5-6 ng/mL by 48 h of age in the calf. The 9,13-di-cis-RA was also a major circulating product of 9-cis-RA dosed intramuscularly to rats; conversely, intravenous administration of 9,13-di-cis-RA produced circulating 9-cis-RA in the rat. 9,13-Di-cis-RA had little or no affinity for cellular retinoic acid binding protein types I and II. This study establishes 9,13-di-cis-RA as a naturally-occurring retinoid under physiological conditions, shows that it undergoes interconversion with 9-cis-RA, and emphasizes a need for careful chromatography to resolve 9-cis-RA and 9,13-di-cis-RA. This is consistent with in vivo 13-cis isomerization operating to modify the concentration and perhaps the activity of 9-cis-RA in vivo.

Reduced ubiquinone plasma levels in patients with liver cirrhosis and in chronic alcoholics.

Ubiquinone (CoQ10 coenzyme) is part of the respiratory chain in mitochondria, and acts as a scavenger in oxidative stress in cell membranes. Ubiquinone is mainly synthesized in the liver and partly derived from the diet; its plasma levels significantly correlate with tissue levels in experimental animals and in pathological states in man. By means of an original high-performance liquid chromatography technique, we measured ubiquinone plasma levels in 10 healthy subjects, in 27 patients with cirrhosis and in 22 chronic alcoholics with normal liver function. Ubiquinone levels were markedly reduced in cirrhosis (0.25 [SD 0.21] microgram/ml vs. 0.92 [0.38] in controls; P < 0.001), without any difference between alcohol- and non-alcohol-related disease. Also, in chronic alcoholics ubiquinone levels were nearly halved (0.49 [0.24]). In cirrhosis, ubiquinone plasma levels significantly correlated with cholesterol (P < 0.05), and with total bilirubin levels (P < 0.01). Our study highlights a remarkable deficiency in ubiquinone levels in patients with cirrhosis and in chronic alcoholics, to which both reduced hepatic synthesis and nutritional defects may contribute.

Microsomal retinoic acid metabolism. Effects of cellular retinoic acid-binding protein (type I) and C18-hydroxylation as an initial step.

This report extends our observation that cellular retinoic acid-binding protein type I (CRABP) serves as substrate for retinoic acid metabolism by testis microsomes. Retinoic acid bound to excess CRABP was metabolized at 70% of the unbound retinoic acid rate with testis microsomes and at the same rates as unbound retinoic acid with kidney and lung microsomes. Chromatography of testis, lung, kidney, and liver microsomal incubations provided two sets of metabolites each, P1 and P2. The composition of P2 was characteristic of the individual tissue. CRABP had modest quantitative affects on P2 composition, but did not affect P2 qualitatively. Retinoids bound to CRABP, isolated from a testis microsomal incubation, consisted of 50% retinoic acid, 32% P1 and 17% P2, suggesting that CRABP may bind retinoic acid metabolites in vivo. The effect of CRABP on the rate of metabolism was retinoid specific. Two major components of P2, 4-hydroxy-retinoic acid and 4-oxo-retinoic acid, when bound to CRABP were metabolized slowly, if at all, by testis microsomes, in contrast to CRABP-bound retinoic acid which had an elimination t1/2 of 40 min. Unbound retinoic acid, 4-hydroxy-retinoic acid, and 4-oxo-retinoic acid had elimination t1/2 values of 35, 40, and 9 min, respectively. Reduced metabolism of CRABP-bound C4-derivatized retinoids suggests pathways of retinoic acid metabolism besides the one initiated by C4-hydroxylation. This was corroborated by identification of 18-hydroxy-retinoic acid as a testis, lung, and liver microsomal retinoic acid metabolite. Ketoconazole inhibited the metabolism by testis microsomes of free and CRABP-bound retinoic acid with IC50 values of 2 and 0.7 microM, respectively, denoting catalysis by cytochrome P-450. These results indicate that cloistering retinoic acid in CRABP, while permitting metabolism, may operate throughout CRABP-expressing tissues as a mechanism of controlling the concentrations of free retinoic acid.

Exogenous CoQ10 preserves plasma ubiquinone levels in patients treated with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

Ubiquinone is a carrier of the mitochondrial respiratory chain which regulates oxidative phosphorylation: it also acts as a membrane stabilizer preventing lipid peroxidation. In man the quinone ring originates from tyrosine, while the formation of the polyisoprenoid lateral chain starts from acetyl CoA and proceeds through mevalonate and isopentenylpyrophosphate; this biosynthetic pathway is the same as the cholesterol one. We therefore performed this study to evaluate whether statins (hypocholesterolemic drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase) modify blood levels of ubiquinone. Thirty unrelated outpatients with primary hypercholesterolemia (IIa phenotype) were treated with 20 mg of simvastatin for a 3-month period (group S) or with 20 mg of simvastatin plus 100 mg CoQ10 (group US). The following parameters were evaluated at time 0, and at 45 and 90 days: total plasma cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, triglycerides, Apo A1, Apo B and CoQ10 in plasma and in platelets. In the S group, there was a marked decrease in total cholesterol low-density lipoprotein-cholesterol and in plasma CoQ10 levels from 1.08 mg/dl to 0.80 mg/dl. In contrast, in the US group we observed a significant increase of plasma CoQ10 (from 1.20 to 1.48 mg/dl) while the hypocholesterolemic effect was similar to that observed in the S group. Platelet CoQ10 also decreased in the S group (from 104 to 90 ng/mg) and increased in the US group (from 95 to 145 ng/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Exogenous CoQ10 supplementation prevents plasma ubiquinone reduction induced by HMG-CoA reductase inhibitors.

The biosynthetic pathway of the CoQ polyisoprenoid side chain, starting from acetyl-CoA and proceeding through mevalonate and isopentenylpyrophosphate, is the same as that of cholesterol. We performed this study to evaluate whether vastatins (hypocholesterolemic drugs that inhibit HMG-CoA reductase) modify blood levels of ubiquinone. Thirty-four unrelated outpatients with hypercholesterolemia (IIa phenotype) were treated with 20 mg of simvastatin for a 6-month period (group S) or with 20 mg of simvastatin plus 100 mg CoQ10 (group US). The following parameters were evaluated at time 0, 45, 90, 135 and 180 days: total plasma cholesterol (TC), HDL-cholesterol, LDL-cholesterol (LDL-C), triglycerides (TG), apo A1, apo B and CoQ10 in plasma and platelets. In the S group, there was a marked decrease in TC and LDL-C (from 290.3 mg/dl to 228.7 mg/dl for TC and from 228.7 mg/dl to 167.6 mg/dl for LDL-C) and in plasma CoQ10 levels from 1.08 mg/dl to 0.80 mg/dl. In contrast, in the US group we observed a significant increase of CoQ10 in plasma (from 1.20 to 1.48 mg/dl) while the hypocholesterolemic effect was similar to that observed in the S group. Platelet CoQ10 also decreased in the S group (from 104 to 90 ng/mg) and increased in the US group (from 95 to 145 ng/mg). This study demonstrates that simvastatin lowers both LDL-C and apo B plasma levels together with the plasma and platelet levels of CoQ10, and that CoQ10 therapy prevents both plasma and platelet CoQ10 decrease, without affecting the cholesterol lowering effect of simvastatin.

Expression of cellular retinoic acid-binding protein (type II) in Escherichia coli. Characterization and comparison to cellular retinoic acid-binding protein (type I).

Cellular retinoic acid-binding protein type II, CRABP(II), has been expressed efficiently in Escherichia coli from the mouse cDNA and compared to E. coli-expressed cellular retinoic acid-binding protein type I, CRABP(I). CRABP(II) had a molecular weight approximately 15,700, a pI of 5.46, an Amax of 350 nm with A350/A280 of 1.8, a fluorescence excitation maximum at 347 nm, and a fluorescence emission maximum at 465 nm (holoprotein). All-trans-retinoic acid and 3,4-didehydro-, 4-hydroxy-, 4-oxo-, 16-hydroxy-4-oxo-, and 18-hydroxy-retinoic acids bind CRABP(II) and CRABP(I) stoichiometrically (Kd values no greater than approximately 10-20 nM). 9-cis-Retinoic acid exhibited saturation binding to both CRABP(II) and CRABP(I) with similar affinities, Kd = approximately 50-70, as did 13-cis-retinoic acid, Kd = approximately 160-240 nM, demonstrating that CRABP(II) and CRABP(I) have similar orders of selectivity for known retinoids. HoloCRABP(II) transferred approximately 70% of its all-trans-retinoic acid to CRABP(I), however, indicating that CRABP(II) has approximately a 3-fold lower affinity for stoichiometrically binding retinoids than CRABP(I). Stern-Volmer plots of both native and denatured CRABP(II) and CRABP(I) were consistent with comparable loci for the tryptophan residues and similar three-dimensional structures. These results suggest that CRABP(II) and CRABP(I), when expressed in vivo in excess of total retinoids, bind several ligands, and functional dissimilarities between the two proteins would not be related to unique preferences for known endogenous retinoids. Rather, CRABP(I) and CRABP(II) may modulate the steady-state concentrations of retinoids to different set points.

Regulation of the Na,K-ATPase activity of Madin-Darby canine kidney cells in defined medium by prostaglandin E1 and 8-bromocyclic AMP.

The role of PGE1 in regulating the activity of the Na+, K(+)-ATPase in Madin Darby Canine Kidney (MDCK) cells has been examined. PGE1 increased the initial rate of ouabain-sensitive Rb+ uptake by MDCK cells, a process that continued to occur over a 5-day period. The increase in the initial rate of ouabain-sensitive Rb+ uptake in MDCK cells treated with PGE1 could be explained by a 1.6-fold increase in the Vmax for ouabain-sensitive Rb+ uptake. The increase in the Vmax for ouabain-sensitive Rb+ uptake observed in MDCK cells under these conditions can be explained either by an increase in the number of active Na+ pumps, or by an increase in the efficiency of the Na+ pumps. Consistent with the former possibility is the observed increase in the number of ouabain binding sites, as well as the increase in Na+, K(+)-ATPase activity in cell lysates obtained from MDCK monolayers treated with PGE1. The involvement of cyclic AMP in mediating these effects of PGE1 on the Na+, K(+)-ATPase in MDCK cells is supported by: (1) the observation of similar effects in 8-bromocyclic AMP treated MDCK monolayers, and (2) a dramatic reduction of the stimulatory effects of PGE1 and 8-bromocyclic AMP on the Vmax for ouabain-sensitive Rb+ uptake, and on the number of ouabain binding sites in dibutyryl cyclic AMP resistant clone 3 (DBr3) (which is defective in cyclic AMP dependent protein kinase activity). PGE1 independent MDCK monolayers exhibit both an increase in the Vmax for ouabain-sensitive Rb+ uptake and an increase in the number of ouabain binding sites in response to 8-bromocyclic AMP. Apparently, the cyclic AMP phosphodiesterase defect in these PGE1 independent cells did not cause cellular cyclic AMP levels to be elevated to a sufficient extent to maximally increase the Na+, K(+)-ATPase activity in these variant cells.