Densitometric Quantitation Research Articles

A validated method for the thin-layer chromatography in situ densitometric quantitation of capsaicinoids in Habanero pepper (Capsicum chinense Jacq.)

A validated method for the thin-layer chromatography in situ densitometric quantitation of capsaicinoids in Habanero pepper (Capsicum chinense Jacq.)

Greenness profile assessment of selective liquid chromatographic methods for determination of a quaternary antimigraine combination along with three of their related official impurities

Two selective, sensitive and environmentally safe LC methods were developed and validated for determination of paracetamol, caffeine, ergotamine tartrate and metoclopramide in coformulated antimigraine tablets along with p-aminophenol, p-nitrophenol and theophylline as officially specified impurities. The first is based on high-performance thin-layer chromatography (HPTLC) coupled with densitometric quantitation. Separation was achieved on HPTLC silica gel 60 F254 plates as stationary phase using ethyl acetate:aqueous ammonium hydroxide solution:glacial acetic acid (10.0:0.4:0.1, by volume) as a developing system followed by scanning of the separated bands at 210.0 nm. The subsequent method depends on HPLC with diode array detection. The LC separation was accomplished on a Scharlau C18 (250 × 4.6 mm, 5 μm) column using a mixture of 20.0 mm sodium dihydrogen phosphate, pH 3.0, adjusted with o-phosphoric acid and methanol, at a flow rate of 1.3 mL/min in a gradient elution program. The separated peaks were detected at 210.0 nm. The proposed methods have been validated and proven to meet the requirements outlined in the International Council for Harmonisation (ICH) guidelines. The greenness profile evaluation was carried out using three tools, namely, the National Environmental Method Index, the Analytical EcoScale and the Green Analytical Procedure Index tool, and a comparative study was then conducted. Successful application of the developed methods for determination of the cited quaternary mixture in Metograine tablets confirms their suitability regarding the analytical performance and ecological impact in quality control assay and impurity profiling purposes.

Stability-Indicating Determination of Tedizolid Phosphate in the Presence of its Active Form and Possible Degradants.

Tedizolid phosphate is an antibiotic prodrug that is metabolized into tedizolid which is used against various resistant bacterial strains. In this study, tedizolid phosphate was subjected to stress degradation conditions, namely, hydrolysis (neutral, acidic and alkaline), thermal, oxidative and photolytic ones. The prodrug was stable toward thermal and photolytic stress conditions, while it showed significant degradation upon applying oxidative and hydrolytic conditions. Two suggested chromatographic methods are described for separation and determination of tedizolid phosphate from the resulted degradation products. The first method is HPLC using Waters Xselect HSS C18 (250×4.6mm, 5μm) analytical column and mobile phase composed of phosphate buffer (50mM, pH6.5):acetonitrile (70:30, %v/v) pumped at flow rate of 1.0mL/min with UV-detection at 300nm. The second method is a TLC coupled with densitometric quantitation, precoated silica TLC-plates as a stationary phase and a mobile phase of methanol:butanol:ethyl acetate:ammonia (33%, w/v) (60:20:20:10,%v/v) were used. The chromatographed plates were scanned at 300nm. The linearity was confirmed over concentration range of 1-100μg/mL and 1-12μg/band for HPLC and TLC-densitometric methods, respectively. Both methods were found to be suitable for determination of tedizolid phosphate in pure form and in its pharmaceutical formulations.

Validation and method comparison of the use of densitometry to quantify monoclonal proteins in canine sera

Densitometric quantitation using serum protein electrophoresis (SPE) is used to monitor monoclonal proteins (M-proteins) in human patients but has not been validated in the dog. Serum globulin concentrations, species-specific radial immunodiffusion (RID), and ELISAs are currently used in veterinary medicine. We aimed to compare four methods that quantify M-proteins using densitometry and biuret protein (dM-protein) measurements. We also validated the best performing method and compared it with the RID and ELISA methods for measuring canine serum M-protein. Serum from six normal dogs and 83 serum samples from 46 dogs with confirmed monoclonal gammopathies were used. A spike and recovery experiment with purified monoclonal IgG and IgM, inter-run and intra-run variability, linearity under dilution, and lower limit of detection were performed. Results of commercial canine RID and ELISA kits for total class-specific immunoglobulin were compared with dM-proteins. The corrected perpendicular drop gating method had <20% error for IgG/γ-globulin and IgM/β-globulin M-protein quantifications. Linearity (r>.99), intra-run CV (1.1%-2.3%), and inter-run CVs (2.0%-3.5%) were acceptable. Correlation between the RID and densitometry results ranged from r=.25 to r=.88, depending on the class. The RID result was greater than that of the biuret total protein in 26/63 (41%) IgA cases. A panel of IgG, IgA, and IgM RIDs failed to correctly identify an IgM paraproteinemia in 6/6 (100%) cases. Densitometry was not comparable with any other tested method. Densitometric quantitation is a valid technique for measuring M-proteins in the β- and γ-globulin regions. Immunotyping via RID using the tested kit does not appear to detect IgM. Densitometry is recommended for measuring M-proteins in canine patients.

DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G™ (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.

Sodium bicarbonate cotransporter NBCe1 affects the growth and motility of prostate cancer cell lines LNCaP and PC3

Prostate cancer is the most common cancer in men. Prostate cancer develops mainly in older men: 6 cases in 10 are diagnosed in men aged 65 or older while it is rare before age 40, according to the report by the American Cancer Society. The average age at the time of diagnosis is ~66. Similar to other cancers, prostate cancer is developed from genetic and epigenetic changes in normal cells, and environmental and systemic stresses also make significant contributions to cancer aggravation. One of such stresses is acidic tumor microenvironments, which is caused by excessive glycolytic cancer cell metabolism, hypoxia, and inefficient blood perfusion. Acidic pH has pleiotropic effects on cancer cell proliferation, clonal evolution/selection and metastasis, and also modulates immune cell functions. The processes of the pH gradient in cancer cells have recently become targets for anti‐cancer therapies.In this study, we examined the effects of acidic pH environments on prostate cancer growth. We focuses on the Na/HCO3 cotransporter NBCe1 (SLC4A4) that moves Na+ and HCO3− into cells and buffers intracellular H+, resulting in extracellular acidification. To examine whether NBCe1 activity is stimulated in proliferating cancer cell lines, we incubated human prostate cancer cell lines LNCaP and PC‐3 cells and human breast cancer cell line MCF7 cells in hypoxic (1% O2, 5% CO2) conditions for 3 days and measured pHi using the fluorescent pH‐sensitive dye BCECF‐AM, and fluorescent images (excitation at 490 and 440 nm; emission at 535 nm) were captured at different time intervals. pH values were generated by the high K+‐nigericin calibration. The solutions contained 100 μM EIPA to inhibit endogenous NHE. After a rapid initial CO2‐induced acidification, the pHi was recovered. The recovery was significantly increased in hypoxic conditions. It is also inhibited by DIDS and S0859, which block most NBCs. In RT‐PCR with the primers that amplify NBCe1 transcripts, the amplification products with the expected size were detected in both prostate cancer cells and breast cancer cells. Next, we performed immunoblot to examine NBCe1 protein expression in cells. Determined by densitometric quantitation of immunoreactive bands and normalization of pixel intensities to β‐actin, the NBCe1 expression in LNCap and PC‐3 cells was increased in hypoxic conditions, whereas the expression in MCF7 remained unaffected. The NBCe1 upregulation in prostate cancer cells was small, but statistically significant (p &lt; 0.05; paired, two‐tailed Student's test; n = 4). To test whether pH changes mediated by the Na/HCO3 transporter affects the growth of prostate cancer cells, we treated cells with the blocker N‐cyanosulphonamide S0859 (50 μM) in normoxic and hypoxic incubations and counted cells 5 days later using a propidium iodide staining protocol. S0859 reduced the number of cells that were proliferated under the hypoxic condition (p&lt;0.05, n=6; unpaired two‐tailed Student t‐test). We also treated cells with higher amounts of the drug and observed more profound inhibition on cell growth (data not shown). These results demonstrate that NBCe1 is the key protein responsible for acidic microenvironments in human prostate cancer.Support or Funding InformationSupported by the Emory Winship Cancer Center Pilot GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human neutrophil peptide-1 affects matrix metalloproteinase-2, -8 and -9 secretions of oral squamous cell carcinoma cell lines in vitro

ObjectivesThe present study aimed to investigate the effect of HNP-1 on the matrix metalloproteinase (MMP)-2, -8 and -9 secretions of two oral squamous cell carcinoma (OSCC) cell lines (UT-SCC-43A and UT-SCC-43B). DesignIn all experiments, the two OSCC cell lines were incubated with graded concentrations (0, 1, 5, and 10μg/ml) of HNP-1 for 24 and 48h. Cell viability was measured using a colorimetric proliferation test and cell death was analyzed with a colorimetric cytotoxicity detection kit. Enzyme activity of MMP-2 and MMP-9 was detected by using gelatin zymography, and molecular weight forms of MMP-8 were determined by Western-blot and a densitometric quantitation method. ResultsBoth cell lines showed a significant increase in LDH toxicity at 24h (UT-SCC-43A: p=0.005 & UT-SCC-43B: p=0.014). Reduced gelatinolytic activities of proMMP-2 were detected in UT-SCC-43B cell line after 24 and 48h of incubation with HNP-1 (1μg/ml: p<0.001, 5μg/ml: p<0.001, and 10μg/ml: p=0.0225). MMP-8 levels of both cell lines decreased at 200–250kDa after 24h of incubation, while after 48h only UT-SCC-43B decreased at 45–50kDa. ConclusionsOur results indicate that HNP-1 suppresses the secretion of MMP-2, -8, and -9 in OSCC cell lines.

Uni-dimensional double development HPTLC-densitometry method for simultaneous analysis of mangiferin and lupeol content in mango (Mangifera indica) pulp and peel during storage

Mango (Mangifera indica) fruit is one of the important commercial fruit crops of India. Similar to other tropical fruits it is also highly perishable in nature. During storage/ripening, changes in its physico-chemical quality parameters viz. firmness, titrable acidity, total soluble solid content (TSSC), carotenoids content, and other biochemicals are inevitable. A uni-dimensional double-development high-performance thin-layer chromatography (UDDD-HPTLC) method was developed for the real-time monitoring of mangiferin and lupeol in mango pulp and peel during storage. The quantitative determination of both compounds of different classes was achieved by densitometric HPTLC method. Silica gel 60F254 HPTLC plates and two solvent systems viz. toluene/EtOAC/MeOH and EtOAC/MeOH, respectively were used for optimum separation and selective evaluation. Densitometric quantitation of mangiferin was performed at 390nm, while lupeol at 610nm after post chromatographic derivatization. Validated method was used to real-time monitoring of mangiferin and lupeol content during storage in four Indian cultivars, e.g. Bombay green (Bgreen), Dashehari, Langra, and Chausa. Significant correlations (p<0.05) between of acidity and TSSC with mangiferin and lupeol in pulp and peel during storage were also observed.

Hormone replacement therapy enhances IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions: a study on postmenopausal monozygotic twin pairs.

MiRNAs are fine-tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co-twin case–control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen-based hormone replacement therapy (HRT) to explore estrogen-dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54–62-years-old monozygotic female twin pairs discordant for HRT (median 7 years). MCF-7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen’s causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR-182, miR-223 and miR-142-3p expressions in HRT using than in their nonusing co-twins. Insulin/IGF-1 signaling emerged one common pathway targeted by these miRNAs. IGF-1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR-182 and miR-223 on IGF-1R and FOXO3A mRNA as well as a dose-dependent miR-182 and miR-223 down-regulations concomitantly with up-regulation of FOXO3A and IGF-1R expression. Novel finding is the postmenopausal HRT-reduced miRs-182, miR-223 and miR-142-3p expression in female skeletal muscle. The observed miRNA-mediated enhancement of the target genes’ IGF-1R and FOXO3A expression as well as the activation of insulin/IGF-1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.

Determination of Antidiabetic Polysaccharides ofOcimum basilicumSeeds Indigenous to Xinjiang of China by High-Performance Thin-Layer Chromatography-UV/Vis-Mass Spectrometry

Summary The seeds of Ocimum basilicum have traditionally been used for the prevention and treatment of a number of diseases in Xinjiang of China. In this study, six polysaccharide extracts were isolated from the seeds of O. basilicum by sequential extraction and purified. After methanolysis, the monosaccharide compositions of the six polysaccharide extracts were analyzed by high-performance thinlayer chromatography (HPTLC) on HPTLC plates silica gel 60 with a mixture of isopropyl acetate–ethyl acetate–methanol–water (5:4:1:0.1, v/v). After derivatization with the aniline diphenylamine o-phosphoric acid reagent, densitometric quantitation was performed by absorbance measurement at 370 or 630 nm. The results revealed that the polysaccharides in O. basilicum seeds consisted primarily of fructose (hRF 80), glucuronic acid (hRF 58), galacturonic acid (hRF 51), rhamnose (hRF 40), xylose (hRF 25), arabinose (hRF 18), and galactose (hR F 9). Xylose, glucuronic acid, and fructose were the three major components found and account for 45, 31, and 21%, respectively. All extracts contained uronic acids, ranged 3 to 24%. An unknown monomeric unit above glucuronic acid was characterized by mass spectrometry (MS) to be a hexuronic acid, and HPTLC–MS proved to be a well suited method for characterization of polysaccharide-based biopolymers and assignment of its monomers. The polysaccharide extracts (aqueous cold, aqueous hot, acidic, and alkaline) showed inhibitor activities of protein tyrosine phosphatase 1B in vitro with half maximal inhibitory concentration (IC50) values of 8.2, 2.2, 70.9, and 0.8 µg mL –1 , respectively. For the first time, a molecular basis was provided to explain the hypoglycemic effect of the seeds of O. basilicum that has been used as antidiabetic adjuvant in traditional Chinese medicine.

SIMULTANEOUS DETERMINATION OF SOME ANTIPROTOZOAL DRUGS IN THEIR BINARY AND TERNARY MIXTURES WITH MEBEVERINE HYDROCHLORIDE IN DIFFERENT DOSAGE FORMS

A simple, precise, sensitive, and accurate TLC-Densitometric method has been developed and validated for simultaneous determination of the antiprotozoal drugs, metronidazole benzoate (METB), and diloxanide furoate (DF) in dimetrol® and furazole® suspensions. Moreover, the method has been optimized to determine metronidazole (MET), DF, and the antispasmodic drug mebeverine hydrochloride (MEH) in dimetrol® tablets using the same solvent system. Developing system consisting of hexane: acetone: triethyl amine (7: 3: 0.6, by volume) was found to be efficient for good chromatographic separation among the studied drugs using pre-activated silica gel 60 F254 TLC plates. Densitometric quantitation was carried out at 254 nm. The method was successfully applied for determination of MET, DF, MEH, and METB in their bulk powders and in their different dosage forms and the results obtained by applying the proposed TLC-Densitometric method showed no significant difference with those obtained by applying the reported methods.

Phosphorylation and Ubiquitination of Degron Proximal Residues Are Essential for Class II Transactivator (CIITA) Transactivation and Major Histocompatibility Class II Expression

Major histocompatibility (MHC) class II molecules are cell surface glycoproteins that present extracellular antigens to CD4(+) T cells and are essential for initiation of the adaptive immune response. MHC class II expression requires recruitment of a master regulator, the class II transactivator (CIITA), to the MHC class II promoter. Post-translational modifications to CIITA play important roles in modulating CIITA mediated transcription of various genes in different cell types. We have previously linked regulation of CIITA to the Ubiquitin Proteasome System (UPS), and we and others have demonstrated that mono-ubiquitination of CIITA dramatically increases its transactivity whereas poly-ubiquitination leads to CIITA degradation. Here we identify three degron proximal lysine residues, Lys-315, Lys-330, and Lys-333, and a phosphorylation site, Ser-280, located within the CIITA degron, that regulate CIITA ubiquitination, stability, and MHC class II expression. Together, these findings contribute to the developing post-translational modification code for CIITA.

HPTLC analysis of tamoxifen citrate in drug-release media during development of an in-situ-cross-linking delivery system

High-performance liquid chromatography (HPLC) has been the most popular choice for analysis of tamoxifen citrate. In this study, single-step isocratic high-performance thin-layer chromatography (HPTLC) on silica gel 60F 254 HPTLC plates with densitometric quantitation at 258 nm was used for separation of tamoxifen citrate from dissolution media constituents. The method was validated for LOD and LOQ, linearity, accuracy, and precision. The calibration function of the analyte was linear in the range 51.5–309.0 ng per band and the correlation coefficient was 0.9992. The limits of detection and quantitation were 25 and 51.5 ng per band. Relative standard deviations were less than 3.0 and 3.1%, respectively, in repeatability and intermediate precision studies and recovery was 96.6–103.2%.

Novel Role of RanBP9 in BACE1 Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation

Accumulation of the amyloid beta (Abeta) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted Abeta generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and Abeta generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced beta-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced Abeta generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease.

Selective Regulation of Heme Oxygenase-1 Expression and Function by Insulin through IRS1/Phosphoinositide 3-Kinase/Akt-2 Pathway

Heme oxygenase 1 (HO-1) is a representative mediator of antioxidants and cytoprotectants against various stress stimuli including oxidants in vascular cells. Intensive insulin treatment can delay the onset and progression of diabetic retinopathy and other vascularopathies, yet little is known about insulin regulation of anti-apoptotic and antioxidant molecules such as HO-1 in vascular cells. Intravitreous injection or in vitro addition of insulin increased HO-1 protein expression in rat retina and in cultured bovine retinal pericytes, retinal endothelial cells, and retinal pigment epithelial cells. In bovine retinal pericytes, insulin induced mRNA and protein expression of HO-1 in a time- and concentration-dependent manner. Using HO-1 promoter analysis, the luciferase reporter assay showed that induction of HO-1 expression by insulin is mediated by additional response elements in the ho-1 promoter gene, which was not responsive to antioxidants. Insulin-induced HO-1 mRNA expression through activation of PI3-kinase/Akt pathway without affecting ERK and p38 MAPK. Overexpression of an adenoviral vector of native IRS1, IRS2, and Akt dominant negative or small interfering RNA transfection of Akt1 and Akt2 targeted gene demonstrated that insulin regulated HO-1 expression via IRS1 and Akt2 pathway, selectively. Further, insulin treatment prevented H(2)O(2)-induced NF-kappaB and caspase-8 activation and apoptosis via the IRS1/PI3K/Akt2/HO-1 pathway in the pericytes. In conclusion, we suggest that the anti-apoptotic properties of insulin are mediated partly by increasing HO-1 expression at transcriptional level via IRS1/PI3K/Akt2 activation, a potential explanation for how insulin is retarding the progression of microvascular complications induced by diabetes.

Identification of An Atypical Paraprotein with Inconsistent M-Spike Quantitation in Four Patients with Waldenstrom's Macroglobulinemia

The densitometric quantitation of the M-spike in patients with a monoclonal gammopathy is a standard method used to monitor diseases such as multiple myeloma and Waldenstrom's macroglobulinemia. Typically, M-spike quantitation shows synchronous parallel trends with other measures of disease status, such as nephelometric immunoglobulin heavy chain measurements, and with clinical condition. However, we report a series of four patients with Waldenstrom's macroglobulinemia and IgM paraproteins with atypical appearing M-spikes that were significantly discordant with the total IgM measurements and clinical condition (Figure 1). Each of these patients had paraprotein bands that were extremely narrow and intense with slight migration from the sample application site (Figure 2). Although similar band morphology may be seen with cryoglobulins, all of these patients had repeatedly negative cryoglobulin tests. Pre-treatment of these samples with beta-mercaptoethanol (BME) to reduce disulfide bonds led to normal-appearing and migrating M-spikes that, when quantitated, gave precise results that were consistent with the total IgM measurements. This technique has permitted continued use of the M-spike to monitor disease progression in these patients, and demonstrates the importance of careful visual inspection of the SPEP gels prior to performing M-spike quantitation in the clinical laboratory. [Display omitted] [Display omitted]

α2 but Not α1 AMP-activated Protein Kinase Mediates Oxidative Stress-induced Inhibition of Retinal Pigment Epithelium Cell Phagocytosis of Photoreceptor Outer Segments

Oxidative stress causes retinal pigment epithelium (RPE) cell dysfunction and is a major risk factor leading to the development of dry-type age-related macular degeneration. Taking pharmacological and genetic approaches, we address the mechanisms by which sublethal oxidative stress inhibits RPE cell phagocytosis. Sublethal oxidative stress dose-dependently inhibited RPE cell phagocytosis of photoreceptor outer segments (POS) and activated AMP-activated protein kinase (AMPK) as determined by increased Thr172 and Ser79 phosphorylation of AMPKalpha and its substrate acetyl-CoA carboxylase, respectively. Similar to oxidative stress, 5-aminoimidazole-4-carboxamide riboside (AICAR), a pharmacological activator of AMPK, inhibited RPE cell phagocytosis of POS in a dose-dependent manner. Inhibition of RPE cell phagocytosis by AICAR was fully reversed by blockade of AICAR translocation into cells by dipyridamole or inhibition of AICAR conversion to ZMP by adenosine kinase inhibitor 5-iodotubercidin. In agreement, AICAR-induced activation of AMPK was abolished by preincubation with dipyridamole or 5-iodotubercidin. Knock-out experiments further revealed that alpha2 but not alpha1 AMPK was involved in RPE cell phagocytosis and that activation of alpha2 AMPK contributed to the inhibition of RPE cell phagocytosis by oxidative stress. Inhibition of RPE cell phagocytosis by activation of alpha2 AMPK was associated with a dramatic increase in acetyl-CoA carboxylase phosphorylation. In comparison, AMPK had no role in oxidative stress-induced breakdown of RPE barrier function. Taken together, reduction in POS load under oxidative stress might direct RPE cells to a self-protected status. Thus, activating AMPK could have therapeutic potential in treating dry macular degeneration.

TLC-densitometric analysis of β-sitosterol in pumpkin seed oil

A densitometric thin-layer chromatographic method has been established for quantitative determination of β-sitosterol in pumpkin seed oil ( Cucurbita pepo L.). Chromatography was performed on silica gel 60 F 254 TLC plates, with toluene-ethyl acetate-glacial acetic acid 15:4:1 ( v/v ) as mobile phase. Densitometric quantitation was performed at 525 nm. The method was validated by determination of linearity (15–750 μg mL −1 ), precision (RSD = 2.36%), and limits of detection (LOD = 0.65 μg per band) and quantification (LOQ = 1.99 μg per band). Average recovery from the pharmaceutical preparation was 96.15%. The method enables simple, rapid, and precise quantitative determination of β-sitosterol in pharmaceutical preparations and can be used for routine quality control.

CXCL12 Activates a Robust Transcriptional Response in Human Prostate Epithelial Cells

CXCL12 is a CXC-type chemokine that plays important roles in hematopoiesis, development, and organization of the immune system and supports the survival or growth of a variety of normal or malignant cell types. Our laboratory recently showed that CXCL12 is secreted by aging stromal fibroblast cells and is a major paracrine factor that specifically stimulates the proliferation of prostate epithelial cells. The current study shows that this CXCL12-mediated proliferative response may be either ERK-dependent or ERK-independent. Moreover, CXCL12 initiates a previously undefined and complex global transcriptional response in prostate epithelial cells. This CXCL12-mediated transcriptional response directly stimulates the expression of genes encoding proteins that are involved in the promotion of cellular proliferation and progression through the cell cycle, tumor metastasis, and cellular motility, and directly represses the transcription of genes encoding proteins involved in cell-cell adhesion and resistance to apoptosis. Thus, CXCL12 may play a major role in the etiology of benign proliferative disease in the context of an aging tissue microenvironment.

P-Rex1 Links Mammalian Target of Rapamycin Signaling to Rac Activation and Cell Migration

Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gbetagamma and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.