Cholesterol Oxidase

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Artificial Neural Network Modeling to Predict the Non Linearity in Reaction Conditions of Cholesterol Oxidase from Streptomyces olivaceus MTCC 6820

Artificial Neural Network Modeling to Predict the Non Linearity in Reaction Conditions of Cholesterol Oxidase from Streptomyces olivaceus MTCC 6820

DOI: 10.4236/jbm.2019.74002 21 Journal of Biosciences and Medicines ANN model was developed with pH, cholesterol concentration, 4-aminoan- tipyrine, crude COX volume and horseradish peroxidase as five model inputs, Table 3. An experimental design matrix was prepared using CCD consisting of 32 experiments, as shown in Table 3. COX activity (U/ml) served as an output of the ANN, gives the quantitative prediction of the increase in enzyme activity when all the five independent variables are optimized. ANN captures the non-linear behavior of the input parameters thus, affecting the output of the system. The enzyme activity is largely affected by the physico-chemical parame- ters of the reaction conditions provided. The above mentioned reaction condi- tions needed to be optimized for a new source of COX from Streptomyces oliva- ceus to predict the optimum activity of COX. The optimum values of assay pa- rameters were found to be: pH of the reaction mixture (8.0), cholesterol concen- tration (0.6% (w/v)), 4-aminoantipyrine (1.5 mM), crude COX volume (100 µl) and horseradish peroxidase (10.0 U/ml), which are different from those reported for cholesterol oxidase activity from other sources of cholesterol oxidase [21] [22]. Maximum cholesterol oxidase activity obtained was 1.1 U/ml (predicted-1.073 U/ml) at optimum levels of parameters, which was very close to the predicted response and is 1.71 times higher than the control. The optimized enzyme activ- ity is the result of a combined effect of these assay parameters as a whole, which shows non-linearity. Hence, ANN modeling was the preferable method.

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Cholesterol Oxidase and Its Applications

Cholesterol Oxidase and Its Applications

COX is used in the treatment of bacterial infections be- cause of its ability to alter the physical structure of the cell membrane due to the conversion of cholesterol into cholest-4-en-3-one. COX is produced by a wide variety of microorganism including some life threatening patho- gens such as Rhodococcus equi, Mycobacterium tuber- culosis and M. leprae [155]. Rhodococcus equi is a com- mon soil-dwelling Gram-positive bacterium that fre- quently infects young horses [127]. Some pathogenic bacteria require cholesterol oxidase to infect their host macrophage, probably because of the ability of choles- terol oxidase to alter the physical structure of the mem- brane by converting cholesterol to cholesten-4-en-3-one [156]. As these enzymes are unique to bacteria, they rep- resent a potential target for a new class of antibiotics. Recently, it has been demonstrated that Alzheimer’s dis- ease β-amyloid selectively oxidizes cholesterol at the C-3 hydroxyl group and catalytically produces 4-cholesten-3- one. Therefore, it mimics the activity of cholesterol oxi- dase [128]. Cholesterol is also known to play a key role in the entry of some viruses. Removal of cellular choles- terol rendered primary cells and cell lines highly resistant to HIV-1-mediated syncytium formation and to infection by both CXCR4- and CCR5-specific viruses. Thus, it ap- pears that cholesterol may be critical to the HIV-1 co- receptor function of chemokine receptors and is required for infection of cells by HIV-1 [129].

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Alzheimer disease β amyloid activity 
mimics cholesterol oxidase

Alzheimer disease β amyloid activity mimics cholesterol oxidase

cholesterol oxidase is a bacterial flavoenzyme that is implicated in the ability of bacteria to induce cellular lysis upon infection of their hosts and to induce thrombus (23). Sequence similarity stud- ies have failed to identify a mammalian counterpart, and choles- terol oxidase is generally regarded as unique to bacteria (24). We also found that levels of 4-cholesten-3-one, the product of choles- terol oxidase activity, were significantly elevated in the postmor- tem brain tissue of subjects with AD compared with age-matched controls; the same result was observed in the brains of Aβ trans- genic mice. These findings support the possibility of direct oxida- tion of cholesterol by Aβ in vivo, which may explain the occurrence of aortic atherosclerosis in Aβ transgenic mice (20). Therefore, AD and atherogenesis may share Cu 2+ -mediated oxidation of choles-

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Fabrication of a Cholesterol Biosensor Based on Cholesterol Oxidase and Multiwall Carbon Nanotube Hybrid Composites

Fabrication of a Cholesterol Biosensor Based on Cholesterol Oxidase and Multiwall Carbon Nanotube Hybrid Composites

A cholesterol biosensor of ChOx/MWCNTs containing multiwall carbon nanotubes (MWCNTs) and cholesterol oxidase (ChOx) has been successfully synthesized on glassy carbon electrode (GCE). The presence of MWCNTs not only enhances the surface coverage (Γ) but also exhibits a promising enhanced electrocatalytic activity to hydrogen peroxide. It is stable, pH-dependent, and electroactive as produced on electrode surface. The average surface coverage (Γ) was estimated about 1.86×10 -11

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Purification and Characterization of Cholesterol Oxidase from Novel Sources Aspergillus awamori and Aspergillus fumigatus

Purification and Characterization of Cholesterol Oxidase from Novel Sources Aspergillus awamori and Aspergillus fumigatus

cholesterol.Under its optimal growth conditions at 37⁰C, pH 7 and 0.1g of cholesterol concentrationCHOx is an enzyme of great commercial value widely employed by laboratories routinely used for the determination of steroid alcohol (cholesterol) in food, blood serum and different clinical samples.Our preliminary work led to the conclusion that both Aspergillus sps might be considered as potentially interesting source of extracellular and membrane bound CHOx for clinical and commercial purposes. A perusal of literature has clearly shown that the existence of oxidase has not been reported from A.awamori and A.fumigatuswhich has also been identified as the hyper producer of cholesterol oxidase in the present study.Therefore, this study has provided a novel source for obtaining abundant amount of cholesterol oxidase to meet the needs of the industrial and medicinal fields.These results demonstrate the novelty of the source of cholesterol oxidase.

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Extracellular Cholesterol Oxidase from Rhodococcus sp.: Isolation and Molecular Characterization

Extracellular Cholesterol Oxidase from Rhodococcus sp.: Isolation and Molecular Characterization

Background: Cholesterol oxidase (CHO) has various clinical and industrial applications. Recently, microbial CHO have received a great attention for their wide usage in medicine. Here, taxonomic characterizations of isolated strain from soil, optimization of the conditions for CHO production and biochemical characterizations of produced CHO enzyme were described. Finally, CHO gene was cloned into a cloning vector. Methods: Various samples were collected and cultivated in a screening medium consisting of cholesterol. For isolation of CHO-producing bacteria, well-grown colonies were inoculated into an optimized medium. Different biochemical and microbiological tests were performed on isolated bacteria to identify their properties. For phylogenic analysis, a partial sequence of l6s rRNA was amplified by PCR using universally conserved primers. A modified method was applied for determination of CHO activity. Then, extracellular CHO activity was assessed under different temperature, pH and cholesterol concentration conditions. Finally, CHO gene was amplified by PCR and cloned into STV28. Results: According to the morphological, cultural and biochemical tests, the isolated bacterium was identified as Rhodococcus sp. strain 501 and deposited in GenBank with accession number FN298676. Results showed that optimum temperature and pH for CHO activity were 35°C and 7.5, respectively. Alignment of nucleotide sequence of CHO gene showed 99% homology with other bacterial CHO genes. Conclusion: Rhodococcus sp. strain 501 produced significant levels of extracellular CHO in an optimized medium for a short period. CHO gene was cloned into cloning vector that can be a valuable tool for better identification and further studies on gene expression. Iran. Biomed. J. 14 (1 & 2): 49-57, 2010

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Isolation and characterization of Cholesterol Oxidase Producing Soil Bacterium and In-vitro Assay of Enzyme

Isolation and characterization of Cholesterol Oxidase Producing Soil Bacterium and In-vitro Assay of Enzyme

Cholesterol oxidase (COD) is an enzyme that converts cholesterol into cholest-4-en-3-one [5] . Many microorganisms such as Nocardia rhodocorus, Arthobacter simplex, Pseudomonas sp, Rhodococcus sp, Streptomyces hygroscopius and Brevibacterium and few other fungal species have been reported to produce COD [7] . COD enzyme has many applications in medicine, agriculture, industry and pharmaceutical purposes. For instance, it can be used for production of diagnostic kits to detect blood cholesterol, biological insecticide and precursors for steroid hormones [5] .The enzyme has been used in the determination of serum cholesterol and in the clinical diagnosis of arteriosclerosis and other lipid disorders [2] . It participate in bile acid biosynthesis.COD is used in the production of precursors of hormonal steroids from cholesterol.COD exhibits potent insecticidal activity that is very important and vital for pest control strategies employing transgenic crops [7] . This enzyme belongs to the family of oxido-reductases, specifically those acting on the CH-OH group of donor with oxygen as acceptor. The systematic name of this enzyme class is cholesterol: oxygen oxidoreductase. Other names in common use include cholesterol- O 2 oxidoreductase, 3beta-hydroxy steroid oxidoreductase, and 3beta-

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Partitioning of Cholesterol Oxidase Produced by Streptomyces olivaceus MTCC 6820 in Different PEG-Salt-Water Aqueous Two Phase Systems

Partitioning of Cholesterol Oxidase Produced by Streptomyces olivaceus MTCC 6820 in Different PEG-Salt-Water Aqueous Two Phase Systems

ABSTRACT: Screening of aqueous two phase systems (ATPS) for partitioning of cholesterol oxidase (Chox) produced by Streptomyces olivaceus MTCC 6820 was done using eight different ATPS composed of two different molecular weight of polyethylene glycol (PEG)X (X= 4000 and 6000) with salts (ammonium sulphate, potassium phosphate, trisodium citrate and sodium sulphate) and water. PEG4000 [10.63% (w/w)] and ammonium sulphate [14.5% (w/w)] was selected as most suitable system components for Chox partitioning on the basis of maximum partition coefficient (K=1.32). 2.34 fold purification of Chox was obtained in the top phase with 75.85% yield and 65.21 U/mg specific activity of partitioned Chox.

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Rapid Identification of Rhodococcus equi by a PCR Assay Targeting the choE Gene

Rapid Identification of Rhodococcus equi by a PCR Assay Targeting the choE Gene

In this work, we studied the usefulness of the R. equi cho- lesterol oxidase gene, choE, recently identified and character- ized in our laboratory (22), as a target for the development of a species-specific PCR method for the rapid identification of this pathogenic nocardioform actinomycete. Routine applica- tion of a PCR-based method requires that the target sequence be highly specific for the microorganism and that it be highly conserved in all strains of that organism. Our results show that the choE target sequence used fulfills these requirements. Se- quences complementary to the COX primers were present in all of the isolates included in a large collection of R. equi strains from different sources and of different geographical origins, suggesting that they are universally conserved in this bacterial species. This is consistent with the production of extracellular choE-derived cholesterol oxidase activity and of its associated phenotypic marker, the CAMP-like reaction with L. ivanovii, by virtually all isolates of R. equi (unpublished data). On the other hand, no choE amplicon was detected in any of the 29 actinomycetes used as negative controls, which included rele- vant pathogenic species, such as N. asteroides and M. tubercu- losis, that also produce cholesterol oxidase activity or that carry choE-related genes (17). Most importantly, these negative con- trol strains included cholesterol oxidase-producing rhodococci, demonstrating the species specificity of our PCR method. The choE gene homologs carried by these bacteria were sufficiently divergent to prevent positive amplification with our COX primers. The specificities of these primers for R. equi were corroborated by sequence analysis of the 16S rDNA from a representative sample of the choE PCR-positive strains.

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Does creatine supplementation improve the plasma lipid profile in healthy male subjects undergoing aerobic training?

Does creatine supplementation improve the plasma lipid profile in healthy male subjects undergoing aerobic training?

To our knowledge, only Earnest et al. [7] reported a spe- cific effect of Cr supplementation on the plasma lipid pro- file. This group observed that physically active subjects with high basal cholesterol levels exhibited a reduction in blood total cholesterol, triglycerides and VLDL-choles- terol, after 12 weeks of Cr supplementation. The authors attributed these changes to a reduction in blood glucose (P = 0.051) brought about by Cr supplementation. Reduced blood glucose was interpreted to indicate an enhancement in insulin sensitivity, which simultaneously could improve the lipid profile if followed by a reduction in fasting insulin levels [7]. It is known from other studies that, changes in glycemia and/or insulinemia may directly affect blood lipoproteins [8-10].

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STUDY OF LIPID PROFILE IN WOMEN INFECTED WITH TOXOPLASMA GONDII IN AL NAJAF GOVERNORATE, IRAQ

STUDY OF LIPID PROFILE IN WOMEN INFECTED WITH TOXOPLASMA GONDII IN AL NAJAF GOVERNORATE, IRAQ

The study was conducted 60 out patients and 30 control women to determine the effect of infected with Toxoplasmosis on levels of cholesterol, HDL, Triglyceride, LDL and VLDL in aborted women infected with Toxoplasmosis in compared with healthy group. Who have visited Al-Zahra hospital and Al-Hakeem Hospital in Al- Najaf governorate during the period from October 2014 till April 2015. The results showed significant decrease (P<0.05) in levels of LDL. While increase in levels of cholesterol, Triglyceride, HDL, and VLDL no levels change in Toxoplasmosis infected women in compared to control group.

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Impact Of Polyphenols On Cholesterol Esterase Inhibition – A Mini Review

Impact Of Polyphenols On Cholesterol Esterase Inhibition – A Mini Review

Cholesterol is one of the vital component of cell membrane is essential for the execution of various functions of a cell. Cholesterol is important for the production of steroid hormones, bile salts, vitamin D, etc., and is derived from the dietary sources and also synthesized endogenously in liver. Hence, uptake, endogenous synthesis and catabolism of cholesterol should be balanced for an effective maintenance of its level in the serum. Therefore, an increase in the serum cholesterol level can cause hypercholesterolemia and prolonged condition leads to the occurrence of various types of cardiovascular diseases. There are various mechanisms to prevent hypercholesterolemia and one such strategy is the inhibition of cholesterol esterase (CEase), an enzyme that hydrolyzes the cholesterol ester to free fatty acid and cholesterol. The inhibition of cholesterol esterase prevents diseases such as hypercholesterolemia, dyslipidemia and related diseases such as atherosclerosis, cardiac arrest, stroke and heart attack. Recent approach is the recruitment of plant based polyphenolics for the inhibition of cholesterol esterase and thereby, the prevention of the above cited diseases can be inhibited by polyphenols from plant sources.

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Interaction of Helicobacter pylori Cell Membrane with Non Esterified Cholesterol and Other Steroids

Interaction of Helicobacter pylori Cell Membrane with Non Esterified Cholesterol and Other Steroids

mones are also expressed in human gastric tissues [33- 36]. Still other studies have revealed the expression of sex hormone receptors in gastric cancer [37-39]. These studies, taken together, indirectly demonstrate that sex hormones exist in the human stomach, an environment that H. pylori colonizes and inhabits for many years. Our group has looked closer at this issue by investigating how the cell membrane of H. pylori interacts with sex hor- mones. In 2009 we found that H. pylori glucosylates not only non-esterified cholesterol, but also various steroid compounds with a 3β-OH group, such as pregnenolone, dehydroepiandrosterone, and epiandrosterone [40]. Yet again, as mentioned earlier, estrone with a 3-OH group was incorporated into the cell membrane of H. pylori but not glucosylated once absorbed [27]. These results indi- cate that the 3β-OH group in a steroid molecule is a cru- cial conformation for glucosylation by the enzymatic action of CGT regardless of the differences of other structures among the steroid compounds.

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Inhibition of diet induced atheroma formation in transgenic mice expressing apolipoprotein E in the arterial wall

Inhibition of diet induced atheroma formation in transgenic mice expressing apolipoprotein E in the arterial wall

expression was relatively low in other tissues as compared with the respective endogenous expression of mouse apoE. They showed no difference in plasma cholesterol levels and lipoprotein profile from controls when fed both normal and atherogenic diets. However, after 24 wk of an atherogenic diet, the formation of fatty streak lesions in proximal aorta was markedly inhibited in transgenic mice as compared with controls. Both lesion area and esterified cholesterol content were < 30% of those in controls. In a tissue cholesterol

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Effect of Nelumbo Nucifera Flowers on Plasma Lipids and Glucose in Young, Middle-Aged and Aged Rats

Effect of Nelumbo Nucifera Flowers on Plasma Lipids and Glucose in Young, Middle-Aged and Aged Rats

In the present study, the level of plasma triglycerides was increased in the middle-aged and aged control rats as compared with young control rats. The elevated plasma triglycerides level observed in aging may be due to the lower activity of heart LPL. LPL, which is an enzyme normally expressed in a variety of tissues, most notably skeletal muscles, myocardium and adipose tissue. In the presence of its cofactor, apolipoprotein C- 11, LPL hydrolyzes triglycerides contained in the triglyceride rich lipoproteins, namely VLDL and chylomicrons. Chylomicrons and VLDL transport triglycerides and cholesterol in the circulation. This results in the release of FFA that are taken up by myocytes for energy production or by adipocytes for energy storage. The increased synthesis of lipids would lead to a greater production and secretion of hepatic VLDL into plasma. This effect would compound the factors leading to the hypertriglyceridemia [32]. Hepatic LPL selectively hydrolyses the VLDL-TG forming partial glycerides and free fatty acids. Extra-hepatic LPL such as in heart is involved in the uptake of TG-rich lipoproteins from the circulation [33]. Administration of N. nucifera decreased the triglycerides levels in aged and middle-aged rats as compared with control rats, may be due to mobilization of fat and normalization the plasma lipids. Lowering of plasma TG concentrations may be attributed to the reduced availability of the precursor FFAs and to enhanced peripheral tissue clearance through increased LPL activity. Flavonoids administered rats showed a reduction in the level of triglycerides by enhanced the activity of lipoprotein lipase in heart of animals [34]. Treatment with N. nucifera decoction showed significant decrease in triglycerides levels [30]. In the present study, the reduction of triglycerides levels elicits hypotriglyceridaemic effect of N. nucifera in different aged rats.

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Physiological properties of beetroot crisps applied in standard and dyslipidaemic diets of rats

Physiological properties of beetroot crisps applied in standard and dyslipidaemic diets of rats

mice with 1% tea catechin [30]. In this work, the appli- cation of a dyslipidaemic diet led to the deterioration of the biochemical indices of serum in rats, reflected in an enhanced total cholesterol level (4 fold) and higher activity of transaminases and at the same time lower HDL cholesterol level. Moundras [24] observed an increase in total cholesterol level in a group of rats fed a diet with cholesterol and significantly higher serum tri- glyceride concentrations. Mandukhail also showed that a dyslipidaemic diet increased serum total cholesterol, total cholesterol-to-HDL-cholesterol ratio and athero- genic index [31]. High fat treatment resulted in oxida- tive stress in rats thus enhanced oxidation of low- density lipoprotein, which plays a key role in the genesis of atherosclerosis. Antioxidants such as betalains are known to efficiently protect against this kind of damage [32]. In our study, we did not observe significant changes in the activity of transaminase in animals fed diets with beetroot crisps in comparison to the control groups. Mekkawy [33] observed its increase in a 30-day experiment with rats fed synthetic and natural dyes, as well as beet red-supplemented diet (0.08 and 0.4 g/kg diet). From the present results, we did not observe a higher antioxidant activity of diets containing beetroot crisps; neither the activity of SOD and GPx enzymes nor the total antioxidant status was significantly chan- ged compared to the control group. Nevertheless, many in vitro studies with betalains from red beets have demonstrated that they possess high antiradical and antioxidant activity [5,7,13,34]. Lu and co-authors [35] found that the oral administration of betalains from red beets (5, 20 or 80 mg/kg BW) in irradiated mice signifi- cantly enhanced the activity of SOD and GPx in the liver, spleen and kidney in a dose-dependent manner. In three different experimental tumour models in mice, Kapadia [36] reported that a very low dose of betanin (0.0025%) from beetroot acts as a potent chemopreven- tive agent.

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Role of PAL and PPO Enzyme Activity in Host Pathogen interaction of Chickpea (Cicer Arietinum L) Root Tissue Infected with Fusarium Wilt

Role of PAL and PPO Enzyme Activity in Host Pathogen interaction of Chickpea (Cicer Arietinum L) Root Tissue Infected with Fusarium Wilt

Polyphenol oxidase has been found in number of plants but higher activity is marked during pathogenic condition. Polyphenol oxidase may indirectly influenced activity of peroxidase by the oxidation of phenol i.e. browning reaction as a part of defense mechanism. Many researchers have also reported similar result, in most of the cases the resistant cultivars WR-315 and JCP-27 revealed higher level of activity while in susceptible cultivars JG-62 and GG-4, the level of activity significantly increased marginally during infection. In the present experiment significantly higher activity in infected plants grown in sick plot also suggested that polyphenol oxidase might be involved in oxidation of phenolics in susceptible cultivar (JG-62). These observations further suggested that PPO activity is actively associated during pre infectional and infectional stages as a part of disease resistance mechanism in response to wilt diseases in root tissues of resistant cultivars grown in sick plot i.e. inoculated with Fusarium oxysporium f.sp. ciceris. Our data are in agreement with the findings made by Shukla and Parameswaran (2004); Chowdhury and Sinha (2000) and Shou-SenYan et al. (2005).

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Cholesterol and ocular pathologies: focus on the role  of cholesterol-24S-hydroxylase in cholesterol homeostasis

Cholesterol and ocular pathologies: focus on the role of cholesterol-24S-hydroxylase in cholesterol homeostasis

24S-hydroxycholesterol formation is not the only mech- anism of sterol removal from neurons. Others oxysterols have been identified. Ingoing flux of 27-hydroxycholesterol from the circulation to the brain and outgoing flow of 7-hydroxy-3oxo-4-cholestenoic acid from the brain to the cir- culation have been measured (Bjorkhem, 2006; Heverin et al., 2005). Cholesterol-27-hydroxylase (CYP27A1) forms primar- ily 27-hydroxycholesterol and secondarily 7-hydroxy-3oxo-4- cholestenoic acid. The exact roles of 27-hydroxycholesterol in the brain are not well understood yet. 27-hydroxycholesterol might influence cholesterol synthesis by activation of LXR- beta (Gilardi et al., 2009). CYP27A1 is expressed in all cellular types of the brain, especially in microglia (Gilardi et al., 2009). In addition, it was reported that neuronal stem cells eliminate cholesterol by CYP27A1 pathway (Milagre et al., 2012) whereas CYP46A1 pathway is regulated by Sp transcription factors during neuronal differentiation (Milagre et al., 2012). These transcription factors are recruited at the cyp46a1 promoter gene level. Few data on cholesterol elim- ination by the inner retina and the RPE are available. No 27-hydroxycholesterol is found in the retina, but its oxida- tion product, 5-cholestenoic acid, is detected at high level, with major inter-individuals variability (by a factor three). Pregnenolone is a metabolite of CYP11A1 and was detected in the retina but at much lower levels than cholestenoic acid (Mast et al., 2011). Although CYP27A1 and CYP11A1 are ex- pressed in various retinal cellular types, CYP46A1 is specific to RGC (Bretillon et al., 2007).

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Promotion of human mesenchymal stem cell osteogenesis by PI3-kinase/Akt signaling, and the influence of caveolin-1/cholesterol homeostasis

Promotion of human mesenchymal stem cell osteogenesis by PI3-kinase/Akt signaling, and the influence of caveolin-1/cholesterol homeostasis

To further test how caveolar lipid rafts affect Akt ac- tivity, we attempted to displace Cav-1 from caveolae by means of cholesterol oxidation. Treatment of cells with CHOD has been reported to cause the reversible reloca- tion of Cav-1 from cell surface caveolae to the Golgi ap- paratus in fibroblasts [44]. We confirmed that treatment with CHOD caused Cav-1 to move out of caveolae in MSCs (Fig. 6a). When this pretreatment was adminis- tered for the duration of osteogenic supplement-induced osteogenesis, matrix mineralization was enhanced (Fig. 6b). Furthermore, when MSCs were pretreated with CHOD, Akt phosphorylation in response to osteogenic supplements was enhanced (Fig. 6c, d). Taken together, our results suggest that Akt activation is important for osteogenic supplement-induced MSC osteogenesis, and

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A Novel Sterol Desaturase-Like Protein Promoting Dealkylation of Phytosterols in Tetrahymena thermophila

A Novel Sterol Desaturase-Like Protein Promoting Dealkylation of Phytosterols in Tetrahymena thermophila

which they were subjected to saponification with 2 ml of 2 M NaOH in methanol- water (1:1, vol/vol) at 60°C for 1 h. Cholesterol or ergosterol was used as the internal standard and was added prior to extraction with hexane at a final concentration of 20 ␮ g/ml. The organic phase was evaporated under nitrogen, and the residue was resuspended in 100 ␮l of N-methyl-N-(trimethylsilyl) triflu- oroacetamide (MSTFA) and then incubated for 30 min at 70°C. The composition of the steryl trimethylsilyl ether derivatives was analyzed by running samples through an HP-5MS (30 m by 0.25 mm by 0.25 ␮ m; Agilent Technologies) in a Hewlett Packard HP 6890 gas chromatograph. The column was temperature programmed at 10°C/min from 100 to 310°C and subsequently held for 10 min at 310°C. MS was carried out using an HP mass selective detector (model MSD 5973) operated at an ionization voltage of 70 eV with a scan range of 50 to 600 atomic mass units (amu). The retention times and mass spectra of all new peaks obtained were compared to those of standards (Steraloids) and those available in the National Institute of Standards and Technology (NIST) mass spectral library. Phylogenetic and sequence analyses. Available C-5(6) sterol desaturase, C-4 sterol methyl oxidase, 25-cholesterol hydroxylase, C-4 sphingolipid hydroxylase, fatty acid hydroxylase, and ␤ -carotene hydroxylase protein sequences were aligned using Clustal W (34). Phylogenetic analyses were carried out by the neighbor-joining method using the program MEGA4, version 4.0.2 (32), with 10,000 bootstrap samplings or by minimum evolution with 5,000 bootstrap rep- licates. Both methods gave very similar tree topologies. All the sequences were retrieved from the UniProtKB database except XP_001017777.1, which was retrieved from the RefSeq database (NCBI).

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