Biomedical subjects

J B German

Publications and source records attributed to J B German.

At least 55 records · Page 3Linked to original sources

PubMed1998

Docosahexaenoic acid accumulates in cardiolipin and enhances HT-29 cell oxidant production.

The objective of this study was to investigate membrane fatty acids for their effects on mitochondrial function in live cells. Mitochondrial potential and oxidant production were measured in human colonic adenocarcinoma (HT-29) cells with membranes enhanced in either oleic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid. Docosahexaenoic acid-enriched cells had increased mitochondrial potential and produced 5-fold more cellular oxidants than did cells enriched with any other fatty acid. Oxidant production in fatty acid-enriched HT-29 cells did not correlate with the degree of unsaturation for total membrane fatty acids. However, there was a strong correlation between the degree of fatty acid unsaturation of cardiolipin, a critical inner-mitochondrial membrane phospholipid, and oxidant production. Cardiolipin acyl composition is known to influence the activity of electron transport complexes, an effect that can increase mitochondrial oxidant production. Docosahexaenoic acid was enriched to 48 mol% of the fatty acids present in HT-29 cell cardiolipin. These results demonstrate the importance of membrane acyl composition to mitochondrial potential and oxidant production in live cells. Additionally, results suggest that docosahexaenoic acid increases cell oxidant production by accumulating in cardiolipin, where its presence alters electron transport efficiency.

Cardiolipins↗

PubMed1997

Portal venous transfusion up-regulates Kupffer cell cyclooxygenase activity: a mechanism of immunosuppression in organ transplantation.

Portal venous transfusions (PVTs) of blood have been shown to induce significant immunosuppression in animal models of organ transplantation. A proposed mechanism of PVT-induced immunosuppression is via alteration of Kupffer cell arachidonic acid metabolism with increased secretion of the suppressive metabolite prostaglandin E2 (PGE2). This study assessed the hypothesis that PVT increases Kupffer cell PGE2 production via up-regulation of Kupffer cell phospholipase A2 (PLA2) as well as constitutive (COX1) and inducible (COX2) cyclooxygenase. Kupffer cells from Lewis rats were harvested 1 hr after PVT with either 1 ml of Wistar-Furth blood, systemic transfusion (SVT), or saline via portal vein (PVSal). After lipopolysaccharide stimulation, 24-hr Kupffer cell supernatant fractions were assayed for PGE2. PGE2 was increased after SVT (1465+/-234 pg/ml) compared with PVSal (597+/-99; P<0.01). PVT increased Kupffer cell PGE2 (5370+/-533; P<0.001 vs. SVT and vs. PVSal) even more substantially. Kupffer cells from PVT-treated rats were then cultured in the presence of inhibitors of PLA2, COX1, or COX2. When Kupffer cells were treated with mepacrine to inhibit PLA2 (5575+/-453 pg/ml), PGE2 production was not different from that by PVSal-treated controls (6467+/-614 pg/ml), but when Kupffer cells were incubated in the presence of the COX1 inhibitor flurbiprofen (3512+/-407 pg/ml) or the COX2 inhibitor nimesulide (2800+/-830 pg/ml), production was decreased 46.7% and 56.7%, respectively, over control activity without added inhibitor. PVT also increased Kupffer cells COX1 and COX2 mRNA as measured by Northern blot. Heart transplants were then performed from Wistar-Furth donors into Lewis recipients at the time of PVT, SVT, PVSal, or PVT + indomethacin (COX1/2 inhibitor). PVT prolonged allograft survival (12.0+/-0.9 days) compared with PVSal (6.3+/-0.3; P<0.01) or SVT (6.3+/-0.3; P<0.04). Indomethacin shortened graft survival when given with PVT (6.5+/-0.3 days). In summary, PVT increased Kupffer cell PGE2 production, up-regulated transcription of Kupffer cells COX1 and COX2 mRNA, and prolonged cardiac allograft survival. COX1/2 inhibition abrogated the effect of PVT. The results indicated that the immunosuppressive effect of PVT may be mediated by up-regulation of Kupffer cell COX1 and COX2. Manipulation of Kupffer cell arachidonic acid metabolism may be useful in augmentation of PVT-induced immunosuppression.

Animals↗

PubMed1997

Disulfide-mediated polymerization of whey proteins in whey protein isolate-stabilized emulsions.

The effects of protein polymerization in whey protein isolate-stabilized emulsions on emulsion properties were investigated. Polymerization, involving intermolecular disulfide bonds between whey proteins adsorbed at the oil-water interface, increased with increasing storage time following emulsion formation. Ageing resulted in increased aggregation of emulsion droplets, emulsion viscosity and susceptibility to creaming but these effects were lower when thiol-disulfide interchange reactions were inhibited by N-ethylmaleimide (NEM). Following heating to 75 degrees C, disulfide-mediated polymerization of whey proteins increased as did droplet aggregation, emulsion viscosity and creaming. While NEM lowered the extent of disulfide-mediated polymerization it did not affect the measured physical properties of the heated emulsions. Non-covalent interactions appeared to be the principal forces leading to aggregation of emulsion droplets but aggregates once formed were stabilized by disulfide bonds.

Animals↗

PubMed1997

Effect of red wine on endothelium-dependent relaxation in rabbits.

1. Published data on the effects of red wine, ethanol and flavonoids on endothelium-dependent relaxation are equivocal. The present study was undertaken to determine the effects of red wine, ethanol and selected flavonoids present in red wine on endothelium-dependent relaxation. 2. Aortic rings from New Zealand White rabbits were set up in organ baths (20 ml) and contracted with noradrenaline (10(-6) mol/l). An attempt was made to elicit dose-dependent relaxant responses to red wine (15, 30, 40, 80 or 120 microliters), ethanol (5.4, 10.8 and 16.2 microliters) and the flavonoids catechin, epicatechin, quercetin and polymeric phenols (10(-7) to 10(-4) mol/l). In some experiments, endothelium-dependent relaxation to cumulative doses of acetylcholine (10(-9) to 10(-6) mol/l) was determined before and after incubating the rings for 15 min with red wine (120 microliters), ethanol (16.2 microliters), quercetin (10(-5) mol/l), catechin (10(-5) mol/l), epicatechin (10(-5) mol/l) and PPs (10(-5) mol/l) respectively. cGMP was also measured in some rings in the control state and after addition of 120 microliters of red wine, sodium nitroprusside (10(-4) mol/l) and polymeric phenols (10(-5) mol/l). 3. Red wine evoked a dose-dependent relaxation in aortic rings. The highest volumes of wine (120 microliters) relaxed the vessels by 71.35 +/- 7.89% of the maximal contraction (8.95 +/- 0.97 g). Polymeric phenols also relaxed the precontracted rings. These responses were abolished by NG-L-arginine methyl ester (L-NAME) and by removal of endothelium. Addition of red wine, polymeric phenols and sodium nitroprusside increased the cGMP content of the rings. In tissues previously incubated with red wine and polymeric phenols, endothelium-dependent relaxation in response to acetylcholine was attenuated. Ethanol had no such effect. 4. Acute exposure of aortic rings to red wine and polymeric phenols evokes an endothelium-dependent relaxation which is mediated by nitric oxide. However, prior exposure to both red wine and polymeric phenols has a second effect in that it attenuates the endothelium-dependent relaxation evoked by acetylcholine. Since this effect is restored by arginine, it is likely to be due to depletion of substrate for nitric oxide synthase.

Animals↗

PubMed1997

Transfection of avian LMH-2A hepatoma cells with cationic lipids.

LMH-2A is an estrogen-responsive avian hepatoma cell line whose susceptibility to cationic-lipid-mediated transfection is poorly described. 3 beta[N-N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DCC) requires a one-step synthesis, and can be used to formulate transfection-grade liposomes when combined with dioleoylphosphatidyl-ethanolamine (DOPE) 1/1 (wt/wt). Luciferase activities in LMH-2A cells were 8.5-fold and 87.5-fold greater than those in HepG2 and FTO2B cells, respectively, following DCC-liposome-mediated transfection with a reporter consisting of the human cytomegalovirus immediate-early promoter (CMV), joined to Photinus pyralis luciferase (L) cDNA, designated pCMVL. Using pCMVL, N-(2-bromoethyl)-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-propana minimun bromide) (BMOP)/DOPE 1/1 (wt/wt), at a 7.5:1 ratio with DNA, produced luciferase activities that were 2.9-fold higher than those of DCC-liposomes, at an optimal 10:1 lipid:DNA ratio. At optimal lipid:DNA ratios, commercially available liposomes, Transfectam, Lipofectamine, and Lipofectin, produced luciferase activities that were 1.39, 1.03, and 0.47-fold those of DCC-liposomes. The effect of 0, 10, 100, or 500 nM/L 17 beta-estradiol on the expression of pCMVL and a second luciferase reporter containing the -593/+48 promoter region of the estrogen-responsive avian apo VLDL-II gene, designated pApoL, was tested in cells cultured in the presence or absence of 10% chicken serum. The CMV promoter supported a high level of expression in LMH-2A cells that was unaffected by serum alone, but was weakly responsive to estrogen. Estrogen responses of both reporters reached a plateau at 10 nM/L. Estrogen increased the expression of pApoL 24-fold and 79-fold in the absence and presence of serum, respectively. The -593/+48 region of the apo VLDL-II promoter may not contain previously reported negative insulin response elements, but chicken serum contains factors that enhance estrogen responsiveness of this region.

Analysis of Variance↗

PubMed1996

Simultaneous determination of multiple aldehydes in biological tissues and fluids using gas chromatography/stable isotope dilution mass spectrometry.

Biological oxidative stress has been associated with various degenerative disorders and disease states, and accurate and sensitive methods are needed to determine the extent of oxidation occurring in vivo. Peroxidation of polyunsaturated fatty acids forms complex mixtures of aldehydes and other breakdown products because various oxidants are involved and lipid composition is not uniform. Quantitative analysis of multiple lipid peroxidation products yields a more complete measure of biological oxidation than measurement of a single aldehyde, particularly when aldehydes exhibit marked differences in reactivity. This report describes extensions of an established gas chromatography/mass spectrometry method to include stable isotope dilution determination in tissue and plasma of three aldehydic products of lipid peroxidation: hexanal, nonanal, and 4-hydroxy-2-nonenal. Use of deuterated internal standards for each analyte improved precision and accuracy compared to a single internal standard for all three aldehydes. Improvements are attributed to differences in extraction and derivatization efficiencies for individual analytes owing to substantial differences in reactivity. Electron ionization of oxime-tert-butyldimethylsilyl derivatives gave greater specificity for detecting all three aldehydes than was possible using electron-capture ionization of O-pentafluorobenzyl oxime derivatives. Exchange of the deuterium label of [2,3-2H]HNE internal standard was determined to be minimal during the analyses.

Aldehydes↗

PubMed1996

Dietary modulation of phospholipid fatty acid composition and lipoxygenase products in mouse lung homogenates.

This study investigated the potential of dietary fats to modulate the arachidonic acid content of mouse lung phospholipids and the formation of lipoxygenase products from arachidonic and eicosapentaenoic acids. Prior to breeding, female mice were fed for five months diets with 10 wt% of either olive oil, safflower oil, fish oil, or linseed oil. The same diets were fed to the females during gestation and to the pups from day 18 to day 42 postpartum. On day 42, the phospholipids were extracted from fresh lung tissue and separated into classes [phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylinositol (PI)] by thin-layer chromatography. Methyl esters of phospholipid fatty acids and unesterified fatty acids were analyzed by gas chromatography. At comparable dietary n-3/n-6 ratios, arachidonic acid was reduced 85 and 75% in lungs from mice fed linseed oil and fish oil, respectively, compared to lungs of safflower oil-fed mice. Dietary fats affected the proportion of arachidonic acid in phospholipids in the order: PE > PC > PS > PI. Following incubation of homogenized lung tissue, the total amount of 12-lipoxygenase products was lowest in lungs from mice fed olive oil, and 12-hydroxyeicosatetraenoic acid was lowest in incubated lungs from mice fed linseed oil. Comparison of the amounts of lipoxygenase substrate fatty acids in the individual phospholipids with the lipoxygenase products suggested that the major substrate pool for the 12-lipoxygenase pathway in mouse lung homogenates was PC.

Animals↗

PubMed1996

Determination of hydroperoxides and structures by high-performance liquid chromatography with post-column detection with diphenyl-1-pyrenylphosphine.

A high-performance liquid chromatographic method, using post-column detection with diphenyl-1-pyrenylphosphine (DPPP), was developed for the quantitative and qualitative determination of isomeric lipid hydroperoxides (OOH). The OOH eluted from a normal-phase column were passed through a photodiode array detector and then mixed with DPPP solution in a reaction coil heated at 80 degrees C. DPPP oxide formed by the reaction with OOH was determined by monitoring the fluorescence intensity at 380 nm and excitation at 352 nm. The conjugated diene OOH (13-cis,trans- and 9-cis,trans-OOH) and nonconjugated OOH (12-cis-trans-and 10-cis,trans-OOH) from photosensitized oxidation of methyl linoleate were determined in a molar ratio of 31:29:19:21, respectively. However, only the two conjugated hydroperoxides were detected by ultraviolet absorption at 234 nm. Further applications were carried out for the determination of OOH of methyl oleate and methyl linolenate. This method proved to be useful for the determination of the OOH containing both conjugated and nonconjugated diene structures.

Chromatography, High Pressure Liquid↗

PubMed1996

Increased hepatic delta 6-desaturase activity with growth hormone expression in the MG101 transgenic mouse.

Growth hormone (GH) has many metabolic effects, but its mechanism(s) of action are not fully understood. We studied the short-term effects of endogenously produced GH on liver delta 6-desaturase activity and adipose and liver lipid fraction fatty acid composition in transgenic mice. MG101 transgenic mice ages 73-114 d received zinc to activate the ovine GH transgene for 7 d. Nontransgenic littermates, used as controls, also received zinc. Liver lipids were fractionated into phospholipids (PL), cholesteryl esters, and triglycerides (TG), and retroperitoneal adipose fractionated into PL and TG for fatty acid analysis. Liver microsomes were assayed for delta 6-desaturase activity. Animals expressing the ovine growth hormone transgene had a 2.5-fold higher liver delta 6-desaturase activity than controls. Arachidonate and docosahexaenoate were significantly higher in liver PL of GH transgenic animals compared to controls, but both were decreased in adipose PL in the GH animals. We conclude that increased production of GH affects both production and organ distribution of highly unsaturated fatty acids. The changes in arachidonate in various lipid pools following transgene expression may mediate the systemic actions of GH.

Adipose Tissue↗

PubMed1996

Diversity of the polar lipids of the food-borne pathogen Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive bacterium that can adapt to high salinity and cold. Because the membrane lipids may play a role in its survival and adaptation, we have examined the polar lipids of L. monocytogenes. Extraction of total lipids from L. monocytogenes yielded 7 +/- 1 mg/mL wet cells. Polar lipids represented 64% of total lipids and contained 9% lipid-phosphorus. Polar lipids were separated into 14 components by two-dimensional thin-layer chromatography. Eight components (88% of polar lipids) contained lipid-phosphorus; among these was one major component (34% of polar lipids). Two other phospholipids were ninhydrin-positive components and accounted for 15% of the polar lipids. Orcinol staining revealed two glyco- or sulfo-lipids accounting for 9% of polar lipids. Five components (4% of polar lipids) were amino components free of phosphorus. The major component contained 46% of its fatty acids as 15:0 anteiso, 24% as 17: 0 anteiso, and 11% as 15:0 iso. The fatty acid profile of the remaining polar lipids was variable, consisting primarily of 16:0, 18:0, 15:0 anteiso, and 17:0 anteiso. Their unsaturation level was < or = 20%; however, the major phosphoaminolipid component was 46% unsaturated. The ratios of 15:0 anteiso/17:0 anteiso and 15:0 anteiso/15:0 iso were similar in all classes, averaging 1.5 and 4.5, respectively. Since the adaptation process to stressful environments involves activation of a membrane transport system for the protectant glycine betaine, the membrane lipids may play a role in enabling transport.

Chromatography, High Pressure Liquid↗

PubMed1996

Dietary Juniperis virginiensis seed oil decreased pentobarbital-associated mortalities among DBA/1 mice treated with collagen-adjuvant emulsions.

The propensity of the fatty acid 5,11,14-eicosatrienoic acid (5,11,14-ETA) to replace arachidonic acid in cell membranes, and its inability to be converted to bioactive eicosanoids, suggest that it may be useful in the treatment of autoimmune disorders. Previously, dietary application of oils extracted from 5,11,14-ETA-rich Platycladus orientalis delayed the onset of autoimmune disease in New Zealand Black mice. To gain more knowledge of the efficacy of this fatty acid toward alleviating immunological disorders, a similar oil was used to examine its effects on collagen-induced arthritis in DBA/1 mice, a model characterized by synovial proliferation and joint infiltration by inflammatory cells. Mice were fed AIN76A diet supplemented with 4% (w/w) of either an oil extracted from the seeds of Juniperis virginiensis (0.4% 5,11,14-ETA); a control oil consisting of equal parts olive, linseed and safflower oils; fish oil (90% fish oil and 10% safflower oil); or safflower oil. Mice were immunized with three injections of collagen-adjuvant emulsions, the first injection was intradermal, and the two subsequent injections were intraperitoneal. Mortalities were recorded following a secondary pentobarbital administration intraperitoneally. Mice from the J. virginiensis group had the lowest mortalities (25%) while safflower oil-fed mice had the highest (59%; p < 0.05). While the J. virginiensis group had the lowest mean CD4/CD8 T lymphocyte ratio, the fish oil group had the highest. These observations suggest that manipulation of eicosanoid production by different dietary lipids had different effects on immune responses, possibly through alterations in T lymphocyte subsets. Hypothetically, a downregulation of prostaglandin E2 release could increase the ratio of T helper 1 to T helper 2 lymphocytes and thereby modulate anaphylactic responses. Also, lowered pro-oxidant status may decrease CD4/CD8 T cell ratios and modify immune function.

Animals↗

PubMed1996

Monoglycerides in membrane systems.

Monoglycerides are used abundantly in food systems, ingested and produced in vivo, and recognized as significant mediators in many biochemical processes, yet their function in various membrane systems is only understood at a hypothetical level. This paper provides a comprehensive review of the effects of monoglycerides in membrane systems from three diverse disciplines: nutrition, food science, and membrane biochemistry. An analysis of the data ranging from feeding studies to physical chemistry is given, detailing the role of these common molecules in biological membranes.

Chylomicrons↗

PubMed1996

Biological effects of dietary arachidonic acid. Introduction.

The intent of this symposium is to assemble current knowledge of the role of arachidonic acid (AA) in the diet to provide a conceptual and mechanistic framework for future research. The principal focus is on the varied biological effects of dietary AA, including opposing effects of n-3 and n-6 polyunsaturated fatty acids (PUFA); regulation of n-6 PUFA metabolism, eicosanoid synthesis and gene expression; the importance of AA in infant nutrition and the contemporary Western diet in general; and the effects of AA on tumor promotion. Through its myriad actions and remarkably ubiquitous presence in cells, AA can be argued to affect every cell of the body. Although the varied molecular events associated with the metabolism of AA have been subjects of intense investigation, the ability of AA in the diet to alter AA levels in cellular membranes is poorly described and is thus the focus of this symposium.

Arachidonic Acid↗

PubMed1996

Monoacylglycerols alter the lipid composition and molecular mobility of phosphatidylcholine bilayers: 13C NMR evidence of dynamic lipid remodeling.

The physical effects of monoacylglycerols (MAG) in small unilamellar vesicles composed of phosphatidyl-choline (PC), triolein, cholesterol, and varying amounts of monopalmitin and monoolein were studied by 13C-NMR. The signal to noise ratio of the carbonyls of PC and triolein were enhanced by the addition of 1,2-di-[1-13C]palmitoylphosphatidylcholine and tri-[1-13C]oleoylglycerol. The linewidths of the carbonyl-13C, choline methyl, olefinic carbon, and terminal methyl resonances were measured digitally from vesicles with 0 to 42 mol % of MAG. Significant increases in the linewidth of carbonyl (P < 0.05), olefinic and terminal methyl carbons (P < 0.01) of vesicles containing 42 mol % monopalmitin indicated that these groups experienced restricted molecular mobility at high monopalmitin concentrations. However, more striking was the apparent displacement of triolein from the surface environment of PC bilayers to an oil-like environment in systems containing only 8 mol % monopalmitin. Displacement of triolein from the surface by monoolein occurred only above 15 mol %. Thus, saturated and monounsaturated monoacylglycerols, natural products of lipoprotein metabolism, dynamically alter both the lipid composition and molecular mobility of lipoprotein surfaces in distinct ways.

Chemical Phenomena↗

PubMed1995

Older plasma lipoproteins are more susceptible to oxidation: a linking mechanism for the lipid and oxidation theories of atherosclerotic cardiovascular disease.

Increases in plasma cholesterol are associated with progressive increases in the risk of atherosclerotic cardiovascular disease. In humans plasma cholesterol is contained primarily in apolipoprotein B-based low density lipoprotein (LDL). Cells stop making the high-affinity receptor responsible for LDL removal as they become cholesterol replete; this slows removal of LDL from plasma and elevates plasma LDL. As a result of this delayed uptake, hypercholesterolemic individuals not only have more LDL but have significantly older LDL. Oxidative modification of LDL enhances their atherogenicity. This study sought to determine whether increased time spent in circulation, or aging, by lipoprotein particles altered their susceptibility to oxidative modification. Controlled synchronous production of distinctive apolipoprotein B lipoproteins (yolk-specific very low density lipoproteins; VLDLy) with a single estrogen injection into young turkeys was used to model LDL aging in vivo. VLDLy remained in circulation for at least 10 days. Susceptibility to oxidation in vitro was highly dependent on lipoprotein age in vivo. Oxidation, measured as hexanal release from n-6 fatty acids in VLDLy, increased from 13.3 +/- 5.5 nmol of 2-day-old VLDLy per ml, to 108 +/- 17 nmol of 7-day-old VLDLy per ml. Oxidative instability was not due to tocopherol depletion or conversion to a more unsaturated fatty acid composition. These findings establish mathematically describable linkages between the variables of LDL concentration and LDL oxidation. The proposed mathematical models suggest a unified investigative approach to determine the mechanisms for acceleration of atherosclerotic cardiovascular disease risk as plasma cholesterol rises.

Aging↗

PubMed1995

Decrease of 12-hydroxyeicosatetraenoic acid production in mouse lungs following dietary oleic anilide consumption: implications for the toxic oil syndrome.

A study was performed to examine the ability of dietary oleic anilide to alter 12-hydroxyeicosatetraenoic acid (12-HETE) production. The structure of oleic anilide, synthesized by reacting oleic acid with aniline, was confirmed by mass spectrometry. The purity of oleic anilide, 75%, was measured by gas chromatography. Oleic acid, which constituted the remaining 25%, is a major component of the rapeseed oil vehicle. Balb/c mice were fed oleic anilide as 0.75% of their diet by weight for three weeks. Their lungs were excised and examined for 12-HETE production in vitro. The 12-HETE levels were significantly (p < 0.01) lower in mice fed oleic anilide than in mice fed the oleic acid control diet. This result illustrates eicosanoid production as a target of fatty acid anilide toxicity. The fatty acid composition, including arachidonic acid, of mouse lungs from both dietary groups was not different. This confirms the availability of substrate for 12-lipoxygenase in both groups. Spleen weights were higher in mice fed oleic anilide than in control mice (p < 0.005). These observations are relevant to immunoregulation and the autoimmune syndromes noted in patients of the Toxic Oil Syndrome (TOS).

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1995

Direct characterization of protein adducts of the lipid peroxidation product 4-hydroxy-2-nonenal using electrospray mass spectrometry.

Oxidative stress and exposures to xenobiotic substances generate reactive substances including the cytotoxic aldehyde 4-hydroxy-2-nonenal. This aldehyde exhibits a variety of biological effects and has been reported as a marker of lipid peroxidation. The toxicity and atherogenicity of 4-hydroxy-2-nonenal have been attributed to the formation of covalent protein adducts. In the current study, two model proteins, beta-lactoglobulin B and human hemoglobin, were exposed to 4-hydroxy-2-nonenal, and the protein adducts were characterized using electrospray ionization mass spectrometry. Our findings provided clear and direct evidence that > 99% of protein modification occurred via Michael addition, and only trace amounts of Schiff base adducts were formed. Confirmation of this result was obtained via quantitative conversion of the modified proteins to oxime and pentafluorobenzyl oxime derivatives as demonstrated by electrospray ionization mass spectrometry, spectrophotometric protein carbonyl assays, and gas chromatography/mass spectrometry determination of 4-hydroxy-2-nonenal released upon treatment with hydroxylamine. These results further demonstrate the availability of the protein-bound aldehyde for subsequent reaction or as a site of molecular recognition. The preponderance of Michael addition products over Schiff base adducts also suggests that most methods for determining 4-hydroxy-2-nonenal in biological tissues or fluids are based on erroneous assumptions that hydrazines or hydroxylamines release 4-hydroxy-2-nonenal from proteins.

Aldehydes↗

PubMed1995

Incorporation of tributyrin enhances the expression of a reporter gene in primary and immortalized cell lines.

Advancing the efficiency of foreign gene transfection can be accomplished by improving its DNA delivery or expression or varying the composition of the DNA construct. A novel method of enhancing gene expression by incorporating triacylglycerol into a transfection protocol is described. The triacylglycerol form of butyric acid was chosen because the addition of the free fatty acid to cells increases gene transcription. The effects of this lipid moiety on transfected gene expression were determined for a range of promoters and cell types. In several cases, inclusion of tributyrin resulted in a greater increase in the production of the gene marker product luciferin than that found for the inclusion of free butyric acid. Overall, the relative effects of tributyrin and free butyric acid differ quite markedly for different cell types and promoters. Thus, tributyrin may be a viable alternative to butyric acid in transfection systems where the free fatty acid has little effect.

3T3 Cells↗