Open Access Short Research Article

Phosphatidylcholine-Mediated Aqueous Diffusion of Cellular Cholesterol Down-Regulates the ABCA1 Transporter in Human Skin Fibroblasts

David Akopian, Ryoko L. Kawashima, Jheem D. Medh

International Journal of Biochemistry Research & Review, Page 214-224
DOI: 10.9734/IJBCRR/2015/14058

ATP-binding cassette protein A1 (ABCA1) is a cholesterol transporter that contributes to the active transport/removal of excess cellular cholesterol. ABCA1 expression is up-regulated when cells accumulate cholesterol. 

Aims: The purpose of this study was to determine any correlation between extracellular phospholipid levels and ABCA1 expression and function. 

Methodology: Human foreskin fibroblasts were incubated with cholesterol alone or cholesterol and phosphatidylcholine. Total RNA was isolated and subjected to end-point RT-PCR to compare ABCA1 transcript levels. Cell lysates were subjected to Western blot analysis to compare ABCA1 protein levels. Cells were loaded with radiolabeled cholesterol and cellular cholesterol efflux was measured in the presence and absence of apoE, a cholesterol acceptor.  ApoE-dependent efflux was calculated as a measure of ABCA1-mediated efflux. 

Results: Here we show that incubation of cholesterol-loaded human skin fibroblasts with L-a-phosphatidylcholine (PC) decreases ABCA1 mRNA and protein levels by 93% and 57%, respectively, compared to cells loaded with cholesterol alone.  Similarly, PC treatment results in a 25% reduction in ABCG1 mRNA levels compared to cells treated with cholesterol alone, but there is no change in SR-BI transcript levels. Subsequent incubation of phospholipid-treated cells with a cholesterol acceptor such as apoE for 24 hours shows a 65% reduction in ABCA1-mediated cholesterol efflux compared to efflux in cells not treated with PC. During the lipid treatment itself, there is a 2.7-fold greater loss of cholesterol from PC treated cells compared to cells treated with cholesterol alone. Measurement of cholesterol in cellular lipid extracts reveals that cells incubated in the presence of phosphatidylcholine are significantly depleted of cholesterol having only 20% of the cholesterol compared to cells loaded with cholesterol alone.

Conclusion: Thus, phosphatidylcholine facilitates removal of cellular cholesterol, thereby negating the cholesterol-dependent induction of ABCA1 message, protein and function.

Open Access Original Research Article

Optimization of Starch and Tryptone Concentrations in Medium Composition for Lipase Production by Submerged Fermentation of Rhizopus arrhizus Using Response Surface Methodology

H. Strinska, V. Dobreva, B. Zhekova, G. Dobrev

International Journal of Biochemistry Research & Review, Page 178-186
DOI: 10.9734/IJBCRR/2015/14439

Aims: The aim of this study is optimization of the concentrations of carbon and nitrogen sources for lipase production by Rhizopus arrhizus using response surface methodology.

Study Design: For this work optimal 22 composite design was used for studying the optimal concentrations of corn starch and tryptone for lipase production by submerged fermentation. A series of planned experiments in three replications was carried out and a mathematical model was developed which was used to describe the process. Optimal levels of studied independent variables were calculated by using the model and the conversion rate.

Place and Duration of Study: This study is a part of PhD dissertation developed in University of Food Technologies, Bulgaria, Department of Biochemistry and Molecular Biology.

Methodology: Maximum lipase activity was achieved by an optimization of some components of the fermentation medium. Corn starch (in concentrations 5.0, 10.0, 15.0 g.dm-3) and tryptone (2.0, 5.0, 8.0 g.dm-3) as independent variables were chosen. Lipase activity was determined by a spectrophotometric assay using synthetic substrate p-nitrophenyl palmitate.

Results: A planned mathematical experiment was carried out and a regression model was developed. The value of R2 was 95.65% which showed that the model had high correlation with the experimental results. The effects of every independent variable had an optimal value while the interaction effects led toenhancement of lipolytical activity. In this case the enzyme activity increased rapidly to 1100 U.dm-3. For lipase activity above that value, large enhancements of the corn starch and tryptone concentrations were needed. In order to use the medium substrates properly there the conversion rate was calculated and it was also considered for the optimization.

Conclusion: By carrying out an optimal composite design a mathematical model was derived, with the aid of which, optimum values of tryptone (6.6 g.dm-3) and corn starch (10.5 g.dm-3) were determined, when the conversion rate and the first derivative of enzyme activity were considered. Those results were confirmed by triplicate experiments at the optimal concentrations. Lipase activity of Ȳ=1340.74 U.dm-3 was achieved, which was very close to the predicted one Ŷ=1235.26 U.dm-3.

Open Access Original Research Article

GC-MS Analysis of Leaf, Fruits and Latex of Croton bonplandianum Baill

Vivekanandadasan Vennila, Rajangam Udayakumar

International Journal of Biochemistry Research & Review, Page 187-197
DOI: 10.9734/IJBCRR/2015/13459

Aims: To characterize the phytocompounds of different parts of Croton bonplandianum using GC-MS.

Study Design: GC-MS analysis of bioactive constituents of C. bonplandianum.

Place and Duration of Study: Post Graduate and Research Department of Biochemistry at Government Arts College (Autonomous), Kumbakonam and Food Safety and Quality Testing Laboratory, Indian Institute of Crop Processing Technology, Thanjavur, Tamilnadu, India, between May, 2011 to June, 2012.

Methodology: The C. bonplandianum leaf and fruit (25 grams) powder was soaked in 60 ml of ethanol each and kept at room temperature for 12 hours and the fresh latex of 10 ml was mixed with 90 ml of ethanol and kept at shaker for 3 hours. The samples were filtered and concentrated. Each sample was subjected to phytochemical analysis using GC-MS.

Results: The GC-MS analysis showed peaks of twenty one phytocompounds from different parts of C. bonplandianum. Out of which five compounds were found in leaf, one compound in latex and fifteen compounds in fruits. The highest peak area of 88.69% for 16-Hexadecanoyl hydrazide and the lowest peak area of 1.39% for Phytol were obtained in leaf extract. The latex of C. bonplandianum showed that the presence of Myo-Inositol, 2-C-methyl with the peak area of 30.8%. The fruits of C. bonplandianum showed that the presence of 9, 12, 15-Octadecatrienoic acid, methyl ester (z,z,z)- with the highest peak area of 41.81% and 2-Hexen-1-ol, 2-ethyl with the lowest peak area of 0.69%.

Conclusion: The phytochemical constituents of C. bonplandianum have been screened and the isolation of individual bioactive compounds from C. bonplandianum will be helpful to find new drugs.

Open Access Original Research Article

Association between Follicle Stimulating Hormone Receptor (FSHR) Polymorphism and Polycystic Ovary Syndrome among Egyptian Women

A. F. Abdel-Aziz, A. M. A. El-Sokkary, A. A. El-Refaeey, H. G. Osman, Rasha A. El-Saeed

International Journal of Biochemistry Research & Review, Page 198-206
DOI: 10.9734/IJBCRR/2015/13896

Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder and the criteria are specified by common complex genetic hyperandrogenism, oligomenorrhea or amenorrhea and polycystic ovary morphology. It is a leading cause of female infertility. The prevelance of PCOS among reproductive age women has been estimated to be 4-12%. The association between PCOS and FSH receptor (FSHR) polymorphism attracts wide attention. The aim of the present study was to evaluate whether polymorphism of FSHR at Ala307Thr codon is associated with PCOS and with clinical features of PCOS patients in Egypt.

Results: PCOS patients (n=64) and control subjects (n=65) in the reproductive age were recruited from the outpatient clinic of Obstetrics and Gynecology Department, Mansoura University, Egypt. The Ala307Thr polymorphism in FSHR, and the frequency of respective genotypes was studied and statistical analysis was performed. We found that the heterozygote Ala/Thr genotype was associated with PCOS (64.1%, OR=2.68, CI=0.97, P= 0.033) compared with controls.

Conclusion: The variant of Ala307Thr polymorphism of FSHR was associated with PCOS but it may be related to high total testosterone levels. In addition the FSHR polymorphism was not associated with either luteinizing hormone or follicular stimulating hormone. The present study suggests that the variant of the FSHR gene may act as a causative factor of PCOS in Egyptian women.

Open Access Original Research Article

Blanching and Juicing Effect on Flavonoids Contents in Commonly Consumed Leafy Vegetables in South West Nigeria

Bamidele Adewale Salau, Kuburat Temitope Odufuwa, Charles Babatunde Adeosun, Adeleke Kazeem Atunnise

International Journal of Biochemistry Research & Review, Page 207-213
DOI: 10.9734/IJBCRR/2015/14230

Blanching and Juicing Effect on Flavonoids Contents in Commonly Consumed Leafy Vegetables in South West Nigeria

Aims: This study investigated the flavonoids level, and effect of blanching and juicing on commonly consumed vegetables in South West Nigeria.

Place and Duration of Study: Chemical sciences, Redeemers University, January to March, 2013.

Methodology: Ten green leafy vegetables were obtained in four major markets in South West Nigeria. Each vegetable was thoroughly mixed and rinsed then divided into three groups, which are fresh, blanched and juice groups with four replicates per group. Flavonoids content were analyzed using standard laboratory methods.

Results: A varying order was observed in flavonoids content of fresh vegetable with highest value observed in Hibiscus esculenta and the lowest in Crassocephalum ruben. Blanching changes the flavonoids content in some vegetables with highest value in Amaranthus viridis and lowest in Basela rubra, while juicing concentrated the flavonoids in some vegetable and at the same time reducing it in some. However, highest value was observed in Hibiscus esculenta and lowest in Colocasia esculenta.

Conclusion: Variation was observed in flavonoids content of fresh vegetables while processing method; blanching and juicing either reduce or increase the flavonoids content.