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Determination of growth-phase dependent influences exerted by prions on yeast lipid content using HPTLC—densitometry

Bui Q., Sherma J., Fried B., Hines K.

Acta Chromatographica

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2016

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Vol. 28, no. 3

373--385

EN

Prions of the baker’s yeast Saccharomyces cerevisiae allow for the inheritance of complex traits based solely on the acquisition of cytoplasmic protein aggregates and confer distinctive phenotypes to the cells which harbor them, creating heterogeneity within an otherwise clonal cell population. These phenotypes typically arise from a loss-of-function of the prion-forming protein that is unable to perform its normal cellular function( s) while sequestered in prion amyloid aggregates, but the specific biochemical consequences of prion infection are poorly understood. To begin to address this issue, we initiated a direct investigation into the potential control that yeast prions exert over fungal lipid content by utilizing the prions [URE3] and [PSI+], the first two prions discovered in yeast. We utilized silica gel high-performance thin-layer chromatography (HPTLC)—densitometry to conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether—diethyl ether—acetic acid (80:20:1) mobile phase, phosphomolybdic acid (PMA) detection reagent]; steryl esters and squalene (hexane—petroleum ether—diethyl ether—acetic acid (50:20;5:1), PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform– diethyl ether—acetic acid (65:25:4.5), cupric sulfate—phosphoric acid) in otherwise clonal prion-infected ([PSI+] or [URE3]) and prion-free ([psi−] or [ure-o]) cells in two growth phases: log-phase and stationary phase. Our analysis revealed multiple statistically significant differences (p < 0.00625) between prion-infected and prion-free cells. Interestingly, prion-induced changes varied dramatically by growth phase, indicating that prions exert differential influences on cell physiology between log and stationary growth. Further experimental replication and extension of the analysis to other prions is expected to resolve additional physiological effects of prion infection. This investigation demonstrates that HPTLC—densitometry is an effective method for studying prion-induced alterations in lipid content in yeast.

2

Evaluation of sample application techniques, stationary and mobile phases, and detection reagents for HPTLC — Densitometry analysis of glucose in fecal samples of mice infected with Echinostoma caproni (Trematoda)

Cicchi M., O’sullivan C. O, Fried B., Sherma J.

Acta Chromatographica

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2011

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Vol. 23, no. 2

295--305

EN

Seven different thin-layer chromatography stationary phases, one additional stationary layer pretreatment, eight mobile phases, two spotting techniques, and three detection reagents were evaluated for the determination of glucose in mouse fecal samples. Quantitative analysis was performed by slit-scanning densitometry. The optimal system was found to be Merck silica gel HPTLC plates with a concentrating zone developed with 1-butanol-glacial acetic acid-diethyl ether-deionized water 27:18:5:3. α-Naphthol-sulphuric acid detection reagent was found to give the best quantitative results, while the naphthoresorcinol reagent was the most useful for qualitative analysis. Semiautomatic application of samples with a CAMAG Linomat was found to give more compact bands and better separations than manual application. Using this system, quantification of glucose was achieved in mouse fecal samples. The amounts of glucose in the fecal samples of BALB/c mice infected with the intestinal trematode E. caproni were compared to control samples of uninfected mice. On the third and tenth days of postinfection, it was determined that the amount of glucose in the infected fecal samples was significantly greater than in the control samples. This indicates that metabolic profiling of glucose using TLC is possible in the mouse model and that TLC may potentially be used to test for the presence of E. caproni in humans.

3

Evaluation of thin-layer chromatography systems for analysis of amino acids in complex mixtures

Vasta J. D., Cicchi M., Sherma J., Fried B.

Acta Chromatographica

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2009

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Vol. 21, no. 1

29-38

EN

Different thin-layer chromatography (TLC) systems were evaluated for analysis of 21 biologically important essential and nonessential amino acids in complex mixtures such as biological tissues and fluids. Amino acids were visualized on the layers by derivatization with ninhydrin reagent, and R F values were determined by slit-scanning densitometry. The five systems found to be most useful for analysis of amino acids were cellulose and silica gel high-performance TLC (HPTLC) plates developed with either 2-butanol-pyridine-glacial acetic acid-deionized water, 39:34:10:26, or 2-butanol-pyridine-25% ammonia-deionized water, 39:34:10:26, and ion exchange TLC plates developed with citrate buffer, pH 3.3. Using these five systems with ninhydrin detection, identification of all amino acids except for leucine and isoleucine in complex mixtures is possible, and quantification can be achieved if the amino acid to be quantified is well separated from adjacent components of the mixture. Example chromatograms are illustrated for separation and identification of amino acids in a snail tissue sample on a cellulose HPTLC plate.

4

Determination of the phospholipid and sphingolipid content in the feces of uninfected BALB/c mice and those infected with Echinostoma caproni by high performance silica gel thin layer chromatography with densitometry

Murray K. E., Fried B., Sherma J.

Acta Chromatographica

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2007

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No. 18

190-198

EN

High performance thin layer chromatography (HPTLC) was used to determine the phospholipid and sphingolipid profiles in the feces of BALB/c mice infected with Echinostoma caproni (Trematoda) versus uninfected controls. Lipids extracted from fecal samples with chloroform-methanol, 2:1, were separated on silica gel HPTLC plates developed in chloroformmethanol- water, 65:25:4. Densitometric analysis of phosphatidylcholine, phosphatidylethanol-amine, and cerebroside zones was performed to compare infected versus uninfected profiles. No significant changes were found in the phospholipid or sphingolipid profiles in the feces of mice at 1 to 7 weeks post infection compared to matched uninfected controls. Although, our findings suggest that fecal polar lipids are not useful biological markers to distinguish E. caproni infections in mice from uninfected controls, the new data presented are important for future comparison in future studies involving feces from mice with heavier levels of infection and urine from infected versus uninfected mice.

5

Thin-layer chromatographic analysis of hydrophilic vitamins in standards and from Helisoma trivolvis snails

Ponder E. L., Fried B., Sherma J.

Acta Chromatographica

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2004

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No. 14

70-81

EN

Separations of the hydrophilic vitamins thiamine (B1), riboflavin (B2), niacin (B3), pyridoxine (B6), cobalamin (B12), ascorbic acid (C), and folic acid have been compared using 14 mobile phases and commercially available, precoated silica gel and chemically bonded silica gel thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) plates. The best separations of individual and mixed vitamin standards was achieved on silica gel plates with 1-butanol–chloroform–acetic acid–ammonia–water, 7:4:5:1:1, benzene–methanol–acetone–acetic acid, 70:20:5:5, and chloroform–ethanol–acetone–ammonia, 2:2:2:1, as mobile phases. Previously reported detection reagents selective for the hydrophilic vitamins were also tested and compared. To determine the usefulness of these chromatographic systems for analysis of biological samples, hydrophilic vitamins in Helisoma trivolvis snails (Pennsylvania strain) were identified and B2 was quantified by videodensitometry. Beca-use of the amount of non-vitamin, water-soluble substances that gave fluo-rescence-quenched zones in the snail extracts analyzed, it was necessary to use natural color or fluorescence or a selective vitamin spray-reagent, in combination with RF values, to identify vitamins in biological samples.

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Thin layer chromatographic analysis of carbohydrates and amino acids in Schistosoma mansoni (Trematoda) cercariae

Wagner S. D., Pachuski J., Fried B., Sherma J.

Acta Chromatographica

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2002

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No. 12

159-169

EN

Thin layer chromatography (TLC) has been used to analyze the carbohydrates and amino acids present in the cercariae of Schistosoma mansoni released from the gastropod Biomphalaria glabrata. Visual observation of the carbohydrate chromatograms showed the presence of glucose and raffinose and three unidentified ?-naphthol-positive zones. Quantification by densitometry of glucose zones in three cercarial samples gave a mean value of 0.491 ng cercaria-1. Raffinose was found to be present at a level of 0.0250 ng cercaria-1 in one sample and below the 0.0200 ng cercaria-1 limit of quantification in two others. Visual observation of the amino acid chromatograms confirmed the presence of histidine, tryptophan, isoleucine, alanine, and proline. Several other unknown ninhydrin-positive zones were also detected. Quantification of the histidine and tryptophan zones in cercarial samples by densitometry gave mean values of 3.71 and 0.588 ng cercaria-1, respectively. The possible functions of carbohydrates and amino acids in the cercariae of S. mansoni are discussed.

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TLC analysis of glucose and maltose in Biomphalaria glabrata (Gastropoda) infected with Schistosoma mansoni (Trematoda)

Schariter J. J., Fried B., Sherma J.

Acta Chromatographica

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2001

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No. 11

102-107

EN

Analysis by thin-layer chromatography (TLC) has been performed on the hemolymph and digestive gland-gonad complex (DDG) of Biompha-laria glabrata snails experimentally infected with Schistosoma mansoni larvae. The major sugars identified in both the hemolymph and DGG of infected and uninfected snails were glucose and maltose. Densitometric quantification showed that there was a significant decrease (Student's t-test, P<0.05) in levels of glucose and maltose in the hemolymph of infected snails 5 and 7 weeks post-infection. At week 7 after infection there was a significant decrease in maltose in the DGG of infected snails. Findings from this work were compared with results previously obtained from spectrophotometric analysis of glucose in B. glabrata infected with larval S. mansoni. It was found that spectrophotometric analysis was less selective and reliable than TLC.

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High-performance thin-layer chromatographic analysis of neutral lipids and phospholipids in Cerithidia californica (Gastropoda) infected with three species of larval trematodes

Cline D. J., Fried B., Sherma S.

Acta Chromatographica

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2000

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No. 10

183-189

EN

High-performance thin-layer chromatography (HPTLC) has been used to analyze neutral lipids and phospholipids in the digestive gland–gonad complex (DGG) of Cerithidia californica snails infected with three species of larval trematode: Euhaplorchis californiensis, Cloacitrema michiganensis, and Mesostephanus appendiculatus. C. californica not infected with larval trematodes were used as control snails. The triacylglycerol content of the DGGs of snails infected with the three species of larval trematodes was significantly less than that of the controls. There was no significant difference between the free sterol content of DGGs infected with any larval trematode type compared with controls. The cholesteryl ester content was significantly lower than that of the controls only in snails infected with C. michiganensis. The situation was variable for free fatty acid content, with M. appendiculatus and E. califor-niensis infections resulting in a significant increase in the concentration of this fraction, compared with that of the controls, and C. michiganensis resulting in a significant decrease. Phosphatidylethanolamine content was increased significantly by all three infections and phosphatidylcholine content was increased by a factor of two or three compared with controls. In conclusion, larval parasitism in C. californica induces different lipid profiles in this marine snail, and these differences can provide chemo-taxonomic information for discrimination between larval trematodes from the snail host.

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TLC and GC-MS identification of glucose and maltose in Biomphalaria glabrata (Gastropoda), and use of quantitative TLC to determine the effect of starvation on the amounts of these carbohydrates

Cline D. J., Fried B., Sherma J.

Acta Chromatographica

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1999

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No. 9

79-86

EN

Maltose and glucose have been identified as the primary carbohydrates in the planorbid snail Biomphalaria glabrata by high performance thin layer chromatography (HPTLC) on silica gel, amino-bonded, C18 reversed-phase, and cellulose plates. The use of specific detection reagents for reducing sugars, spiking experiments, and differential coloration of sugar zones with ?-naphthol reagent on amino plates aided the identification of glucose and maltose. Gas chromatography–mass spectrometry (GC–MS) analysis confirmed the identification maltose and glucose on the basis of retention times and spectral fingerprints. The presence of trehalose was not confirmed. After identifying maltose and glucose, snails were starved for twelve days and the effects on the concentrations of these sugars were determined. Amounts of glucose dropped significantly in the digestive gland–gonad complex (DGG) from day 0 to day 12; maltose in the hemolymph and DGG and glucose in the hemolymph did not change significantly during the starvation period.