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PubMed2000

Determination of the fatty acid composition of saponified vegetable oils using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) for the determination of the fatty acid composition of vegetable oils is described and illustrated with the analysis of palm kernel oil, palm oil, olive oil, canola oil, soybean oil, vernonia oil, and castor oil. Solutions of the saponified oils, mixed with the matrix, meso-tetrakis(pentafluorophenyl)porphyrin, provided reproducible MALDI-TOF spectra in which the ions were dominated by sodiated sodium carboxylates [RCOONa + Na]+. Thus, palm kernel oil was found to contain capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, and stearic acid. Palm oil had a fatty acid profile including palmitic, linoleic, oleic, and stearic. The relative percentages of the fatty acids in olive oil were palmitoleic (1.2 +/- 0.5), palmitic (10.9 +/- 0.8), linoleic (0.6 +/- 0.1), linoleic (16.5 +/- 0.8), and oleic (70.5 +/- 1.2). For soybean oil, the relative percentages were: palmitoleic (0.4 +/- 0.4), palmitic (6.0 +/- 1.3), linolenic (14.5 +/- 1.8), linoleic (50.1 +/- 4.0), oleic (26.1 +/- 1.2), and stearic (2.2 +/- 0.7). This method was also applied to the analysis of two commercial soap formulations. The first soap gave a fatty acid profile that included: lauric (19.4% +/- 0.8), myristic (9.6% +/- 0.5), palmitoleic (1.9% +/- 0.3), palmitic (16.3% +/- 0.9), linoleic (5.6% +/- 0.4), oleic (37.1% +/- 0.8), and stearic (10.1% +/- 0.7) and that of the second soap was: lauric (9.3% +/- 0.3), myristic (3.8% +/- 0.5), palmitoleic (3.1% +/- 0.8), palmitic (19.4% +/- 0.8), linoleic (4.9% +/- 0.7), oleic (49.5% +/- 1.1), and stearic (10.0% +/- 0.9). The MALDI-TOFMS method described in this communication is simpler and less time-consuming than the established transesterification method that is coupled with analysis by gas chromatography/mass spectrometry (GC/MS). The new method could be used routinely to determine the qualitative fatty acid composition of vegetable oils, and, when fully validated by comparison with standard analytical methodologies, should provide a relatively fast quantitative measurement of fatty acid mixtures and/or soap formulations that contain saturated and unsaturated hydrocarbon moieties.

Esterification↗
PubMed2002

Isolation and structure elucidation of an isoflavone and a sesterterpenoic acid from Henriettella fascicularis.

A new isoflavone, 4',5,7-trihydroxy-6,8-dimethylisoflavone (1), and a new sesterterpenoic acid (2), together with five known compounds, lichexanthone (3), (-)-pinoresinol (4), betulinic acid, palmitic acid, and beta-sitosterol, were isolated from a dichloromethane extract of the branches of Henriettella fascicularis. Their structures were established by extensive spectroscopic methods. An attempt to determine the absolute stereochemistry of (2E,6S)-6-[(1R,5Z,3aS,9R,10Z,12aR)-1,2,3,3a,4,7,8,9,12,12a-decahydro-9-hydroxy-3a,6,10-trimethylcyclopentanocycloundecen-1-yl]-2-methylhept-2-enoic acid (2) was performed by single-crystal X-ray analysis, using Cu Kalpha radiation. Compound 1 showed significant competitive binding to estrogen receptor beta and moderate antiestrogenic activity with cultured Ishikawa cells.

Alkaline Phosphatase↗
PubMed1979

Purification of Streptomyces chromofuscus phospholipase D by hydrophobic affinity chromatography on palmitoyl cellulose.

Phospholipase D [phosphatidylcholine cholinehydrolase, EC 3.1.4.4] excreted from Streptomyces chromofuscus was purified from the culture supernatant by precipitation with acetone and column chromatographies on palmitoylated gauze (Pal-G), DEAE-cellulose, and Sephadex G-150 with an overall recovery of 46% and 1000-fold increase in specific activity. The purified enzyme preparation showed a single band on sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. The enzyme had a molecular weight of about 50,000 by gel filtration on Sephadex G-150 or about 57,000 by SDS-polyacrylamide disc gel electrophoresis and an isoelectric point (pI) of pH 5.1 on isoelectric focusing. The enzyme hydrolyses lecithin, lysolecithin, sphingomyelin, and cephalin; the relative reaction velocities and Km's for choline-phospholipids were 87% and 1.43 mM for lecithin, 100% and 1.67 mM for lysolecithin, and 22% and 0.56 mM for sphingomyelin. The enzymatic reaction was optimal at pH 8, and its velocity was appreciably increased by either detergent (Triton X-100, deoxycholate), Ca2+ or both detergent and Ca2+. Diethyl ether stimulated the enzymatic activity by 30%; SDS and EDTA inhibited the activity. Bovine serum albumin, Triton X-100, and lipids (lecithin, lysolecithin, phosphatidic acid, lysophosphatidic acid, palmitic acid, and oleic acid) inhibited adsorption of the purified enzyme onto palmitoyl cellulose (Pal-C) and affected both the enzyme activity and stability: albumin and Triton X-100 increased the activity and enhanced the heat-stability; lysophospholipids decreased the activity but other lipids increased the activity; all the lipids lowered the heat-stability. The enzyme adsorbed on Pal-C was active, although its activity was about one-ninth of that of free enzyme, and was protected from heat-inactivation. Thus this enzyme appears to possess a hydrophobic site distinct from its catalytic site and to be adsorbed onto Pal-C through the hydrophobic site. Albumin, Triton X-100, and lipids seem to bind to the hydrophobic site and to have an appreciable effect on the enzyme activity and stability.

Cations, Divalent↗
PubMed2002

Antiproliferative effects of a non-beta-oxidizable fatty acid, tetradecylthioacetic acid, in native human acute myelogenous leukemia blast cultures.

The lipid metabolism is important in the regulation of cell proliferation. We have examined effects of a fatty acid analogue, tetradecylthioacetic acid (TTA), on the functional phenotype of native, human AML cells. TTA inhibited AML blast proliferation in the presence of single cytokines (GM-CSF and SCF: P > 0.05, 35 patients with detectable proliferation) and a combination of cytokines (P < 0.005, n = 21). This antiproliferative effect was generally stronger than for the normal fatty acid palmitic acid (PA). Both TTA and PA increased the secretion of tumor necrosis factor alpha (TNFalpha) (P < 0.05, 27 patients with detectable cytokine release), but only PA increased interleukein 1beta (IL-1beta) release (P < 0.005, n = 34). AML blast populations varied significantly in their levels and activities of metabolites and enzymes characterizing oxidative status and fatty acid metabolism, and there was no significant correlation between the intrinsic oxidative status and the effects of PA and TTA on blast proliferation. Although TTA reduced the proliferation of mitogen-stimulated normal T cells derived from healthy individuals (P < 0.05, n = 8), no adverse effects were seen on peripheral blood cell counts (reticulocytes, platelets, total white blood cells, differential leukocyte counts) for healthy volunteers receiving TTA (oral administration of 1000 mg/day for 7 consecutive days). Our results suggest that TTA can inhibit AML blast proliferation through pathways that are unrelated to autocrine cytokine secretion and intrinsic oxidative status.

Acute Disease↗
PubMed1990

Neonatal exposure to DDT and its fatty acid conjugate: effects on cholinergic and behavioural variables in the adult mouse.

We have recently observed that DDT and a DDT metabolite, DDOH, conjugated to a fatty acid, palmitic acid, DDOH-PA, affects muscarinic cholinergic receptors (MAChR) in the neonatal mouse brain when given to suckling mice during rapid brain growth. This early exposure of the neonatal mouse also affects the behaviour of the animals as adults. When DDT and DDOH-PA was given as a single low oral dose of 1.4 mumol/kg body weight, DDT (0.5 mg), DDOH-PA (0.7 mg) and a 20% fat emulsion vehicle (10 ml) per kg body weight to 10-day-old NMRI mice, behavioural tests at adult age of four months, indicated disruption of a simple, non-associative learning process, i.e. habituation, in both DDT and DDOH-PA treated mice. There was also a significant increase in the potassium evoked release of ACh from slices of cerebral cortex and a tendency towards a decrease in the density of MAChR in mice receiving DDT. These effects in the adult mice could not be correlated to the concentration of DDT in the adult brain since DDT one month after its administration to the 10-day-old mouse no longer is present in the brain.

Animals↗
PubMed2003

Discrimination against 13C during degradation of simple and complex substrates by two white rot fungi.

Changes in isotopic 13C signatures of CO2-C evolved during decomposition of a sugar (glucose), a fatty acid (palmitic acid), a protein (albumin), a structural biopolymer (lignin) and bulk plant tissue (aerial shoots from Lolium perenne) were monitored over a period of 76 days. All materials were sterilized and inoculated with either of two different species of white rot fungi, Phanerochaete chrysosporium or Coriolus versicolor, and incubated in sealed bottles at 28 degrees C. The CO2 concentration in the jars was periodically determined using an infrared gas analyzer and its isotopic (13C) signature was assessed using a trace gas (ANCA TGII) module coupled to an isotope ratio mass spectrometer (IRMS, Europa 20-20). L. perenne material inoculated with C. versicolor showed the highest C mineralization activity with approximately 70% of total C evolved as CO2 after 76 days of incubation, followed by glucose. Substrates inoculated with C. versicolor generally decomposed faster than when degraded by P. chrysosporium, except for lignin, where no significant differences between the two fungi types were found and CO2-C released was less than 2% of the initial C. Considerable 13C isotopic fractionation during the degradation of plant tissue and of pure biochemical compounds was revealed as well as progressive shifts in cumulative CO2-13C isotopic signatures over time. During the first stages of decomposition, the CO2-C released was usually depleted in 13C as compared with the initial solid substrate, but with ongoing decomposition the CO2-C evolved became progressively more enriched in 13C. P. chrysosporium usually showed a slightly higher 13C fractionation than C. versicolor during the first decomposition phase. At posterior decomposition stages isotopic discrimination was often stronger by C. versicolor. These findings on isotopic 13C discrimination during microbial degradation both of simple biochemical compounds and of complex vegetal tissue confirmed not only the existence of significant 13C isotopic fractionation during plant residue decomposition, but also the existence of non-random isotopic distribution within substrates. They also demonstrated the ability of microorganisms to selectively discriminate against 13C even when degrading an isolated simple substrate.

Albumins↗
PubMedDate not supplied

In vitro and in vivo study of octacosanol metabolism.

BACKGROUND: Policosanol is a mixture of very-long-chain aliphatic alcohols purified from sugar cane wax with cholesterol-lowering effects, whose main component is octacosanol. Scarce data about the metabolism of octacosanol and the other fatty alcohols composing policosanol have been published. METHODS: Human fibroblasts were cultured in presence of (3)H-octacosanol during 0.5, 2 and 4 h. Lipid extracts were analyzed by thin layer chromatography, and the spots corresponding to octacosanol and octacosanoic acid were identified comparing with authentic standards. Spots were scraped, transferred to vials and radioactivity was measured. For corroborating the presence of octacosanol and octacosanoic acid, samples were analyzed by gas chromatography-mass spectrometry (GC-MS). The in vivo study of octacosanol metabolism was conducted in rats and Macaca arctoides monkeys. Rats were orally administered with policosanol (60 mg/kg) and free octacosanol and octacosanoic acid were identified in liver and plasma by GC-MS at various time intervals. Monkeys were orally and endovenously treated with policosanol (10 mg/kg) and the presence of free octacosanol, octacosanoic acid and some chain-shortened FA was investigated. RESULTS: When fibroblasts were cultured in presence of (3)H-octacosanol, three spots were found: a first one corresponded to octacosanoic acid, a second to octacosanol and a third one remained unidentified. The radioactivity on the spot of octacosanoic acid slightly decreased throughout the incubation but increased in the third spot. Octacosanol and free octacosanoic acids were also identified in plasma of monkeys orally administered with policosanol. In addition, plasma samples showed free saturated acids, palmitic acid being the most abundant, followed by oleic and mystiric acids. Unsaturated acids (oleic and palmitoleic) were also observed. CONCLUSIONS: The present study demonstrates that octacosanoic acid is formed after incubation of fibroblast cultures with (3)H-octacosanol and after oral dosing with policosanol to rats. In addition, we demonstrated that shortened saturated (myristic, palmitic and stearic) and unsaturated (oleic, palmitoleic) FA are also formed after oral dosing with policosanol to monkeys. The present results are consistent with the fact that octacosanol metabolism is linked to FA metabolism via beta-oxidation, but further studies need to explore the occurrence of more metabolites proving such hypothesis.

Animals↗
PubMed1969

Very low density lipoproteins in intestinal lymph: role in triglyceride and cholesterol transport during fat absorption.

The role of nonchylomicron very low density lipoproteins (VLDL, S(f) 20-400) in the transport of triglyceride and cholesterol was studied during lipid absorption. Various long chain fatty acids were infused intraduodenally in the form of mixed fatty acid-mono-olein-taurocholate micelles; control animals received saline or taurocholate. As compared with controls, all fatty acids (palmitic, oleic, linoleic) resulted in significant increases in chylomicron (S(f) > 400) triglyceride. In addition, palmitic acid resulted in a twofold increase in VLDL triglyceride, whereas with the absorption of oleic or linoleic acid VLDL triglyceride did not change significantly. Differences in triglyceride fatty acid composition between chylomicrons and VLDL were observed during lipid absorption. Although the absolute amount of endogenous cholesterol in intestinal lymph was not significantly affected by lipid absorption under these conditions, its lipoprotein distribution differed substantially among the lipid-infused groups. During palmitate absorption, VLDL cholesterol was similar to that in the taurocholate-infused controls, and was equal to chylomicron cholesterol. In contrast, during oleate and linoleate absorption the VLDL cholesterol fell markedly, and was less than half of the chylomicron cholesterol in these groups. The half-time of plasma survival of VLDL cholesterol-(14)C was found to be twice that of chylomicron cholesterol-(14)C. These studies demonstrate that dietary long chain fatty acids differ significantly in their effects upon the transport of triglyceride and cholesterol by lipoproteins of rat intestinal lymph. These findings, together with the observed differences in rates of removal of chylomicrons and VLDL from plasma, suggest that variations in lipoprotein production at the intestinal level may be reflected in differences in the subsequent metabolism of absorbed dietary and endogenous lipids.

Animals↗
PubMed2003

A method for determining two substrates binding in the same active site of cytochrome P450BM3: an explanation of high energy omega product formation.

A number of enzymes from the cytochrome P450 family show atypical (non-Michaelis-Menten) kinetic behavior resulting from substrate activation, inhibition, partial inhibition, biphasic saturation, or autoactivation. Herein, we provide a technique that can identify multiple substrate occupancy in the same active site of a P450 as a result of an altered kinetic profile. Using an isotope effect on product ratios confirms that the enzyme-substrate (ES) complex responsible for omega hydroxylation of palmitic acid (palmitate) is in rapid equilibrium with the ES complex that leads to omega-1 hydroxylation of palmitate. Co-incubation of a second substrate, lauric acid (laurate), results in a change in the ratio of omega to omega-1 hydroxylated palmitate. Furthermore, an isotope effect on palmitate is observed when deuterated laurate is co-incubated with non-deuterated palmitate. These results are only consistent with both substrates being in the same active site simultaneously. This mode of binding explains how the F87A mutant of P450BM3 is able to produce the omega alcohol, a product that arises from the high-energy primary radical.

Bacterial Proteins↗
PubMed1992

Albumin and fatty acid effects on the stimulated production of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) by human polymorphonuclear leukocytes.

Production of platelet-activating factor (PAF) during opsonized zymosan stimulation of human polymorphonuclear leukocytes is dependent on the concentration of extracellular albumin and on the presence of exogenous fatty acids. Fatty acid-free albumin caused a concentration-dependent increase in PAF synthesis up to 5% albumin concentrations (w/v) where the amount of PAF produced was three- to four-fold higher than in controls containing no albumin. The addition of free fatty acids, particularly arachidonic acid and palmitic acid, to 5% fatty acid-free albumin media caused a concentration-dependent decrease in PAF synthesis. A 50% inhibition of PAF synthesis was observed at an arachidonic acid concentration of 120 microM and at a palmitic acid concentration of 100 microM. The inhibition of PAF production by palmitic acid was also dependent on the concentration of extracellular albumin. In 0.5% fatty acid-free albumin media, a palmitic acid concentration of 40 microM produced a 50% inhibition in PAF synthesis. The addition of palmitic acid did not affect the release of endogenous arachidonic acid during stimulation. In contrast, the addition of stearic acid up to 120 microM in 5% fatty acid-free albumin media had no effect on PAF production. The different inhibitory effects of palmitic acid and stearic acid on PAF production may be related to differences in intracellular utilization of these two fatty acids during cell stimulation.

Fatty Acids, Nonesterified↗
PubMed1988

Characterization of distinct layers of the Mycobacterium avium envelope in respect of their composition by fatty acids, proteins, oligosaccharides and antigens.

The distribution of fatty acids, proteins, polysaccharides and antigens in subcellular fractions of Mycobacterium avium is described. Significant qualitative differences in the chemical composition of the various fractions have been used to further characterize the tripartite structure of the cell wall. In the outer dense layer (POL), in addition to previously described complex amphiphatic lipids, new oligosaccharides (lipooligosaccharides?) and a major glycoprotein were located; and it was found that tuberculostearic acid (TSA) esterified the phospholipids of this outerlayer. Judging from the data, it was proposed that the phospholipids formed a basic matrix monolayer in which other compounds of the POL intercalated. It was suggested that in an aqueous environment the hydrophobic ends of the phospholipids oriented to face the mycolic acid residues of the cell wall skeletons (or CWS) to form the 12 nm thick electron transparent layer. The purified CWS contained alpha-, keto-, and dicarboxylic mycolic acids; alanine, glutamic acid and diaminopimelic acid; and arabinose and galactose. Two additional nonidentified amino acids and an unidentified sugar were found in the CWS. Also, in the CWS the fatty acids: palmitic acid (21.8%), oleic acid (4.3%), stearic acid (9.2%) and TSA (4.3%), were detected. The main fatty acids detected in the cytoplasmic membrane (CM) were palmitic (20%), oleic (14.5%) and stearic (8.6%) acids. Mycolic acids and TSA were absent in the CM phospholipids. The major proteins of the CM (86, 40, and 26 Kd proteins) were distinct from the major proteins detected in the cytosol (CYT) fraction (43, 36, and 19 Kd proteins). A 58 Kd protein was present in both the CM and the CYT. The CYT and CM antigens were found absent in surface antigens extracted using sodium dodecyl sulphate (SDS).

Antigens, Bacterial↗
PubMedDate not supplied

Time-of-flight secondary ion mass spectrometry can replace histochemistry demonstration of fatty acids in the retina.

PURPOSE: The purpose of this study was to demonstrate fatty acids in the rat retina and to show that time-of-flight secondary ion mass spectrometry (TOF-SIMS) can replace histochemistry. METHODS: Five normal 3-month-old Wistar Kyoto rats were used. The eyes were enucleated under pentobarbital anesthesia, immediately put into liquid nitrogen without any fixation, then cut into semithin sections, which were covered by OCT compound. Serial semithin sections were cut to 8- micro m thickness with a cryoultramicrotome and air-dried. They were placed on a silicon plate and air-dried for 1 h for analysis with TOF-SIMS. Before the specimens were analyzed, the molecular weight of the secondary ion for each molecular substance was measured with the use of commercial samples. These values were used for calculating the count of the secondary ion for each molecular substance. The molecular weights of the secondary ions of maleic acid, palmitic acid, oleic acid, stearic acid, arachidonic acid, and docosahexaenoic acid were determined in the photoreceptor cells. RESULTS: Of the fatty acids in the photoreceptor cells, maleic acid had the highest count, followed in descending order by palmitic, oleic, docosahexaenoic, stearic, and arachidonic acids. CONCLUSIONS: TOF-SIMS can demonstrate the presence of fatty acids in the retina, replacing histochemical techniques.

Animals↗
PubMed1997

Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nuclease-resistance properties.

We report here the site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides (ODNs). ODNs containing both 5-( N-aminohexyl)-carbamoyl-2'-deoxyuridine (H), which serves as a tether for the further conjugation of functional groups, and 5-(N,N-dimethylaminohexyl)carbamoyl-2'-deoxyuridine (D), which contributes to the thermal stability of the duplex and to the resistance to nucleolytic hydrolysis by nucleases, were synthesized. Functional groups such as folic acid and palmitic acid were site-specifically introduced into the terminus of the aminohexyl-linker of H. The thermal stability and resistance toward nuclease digestion of the modified ODNs were studied. We found that ODNs containing D and H formed stable duplexes with both the complementary DNA and RNA strands even when a bulky functional group such as folic acid, palmitic acid or cholesterol was attached to the terminus of the amino-linker. We also found that ODN analogues which contained D were more resistant to nucleolytic degradation by exo- and endonuclease than the unmodified ODN. Furthermore, duplexes formed by ODNs containing D and the complementary RNA could elicit RNase H activity.

Animals↗
PubMed1984

Acylation of plant acyl carrier proteins by acyl-acyl carrier protein synthetase from Escherichia coli.

The acyl-acyl carrier protein synthetase from Escherichia coli has been examined for its ability to specifically acylate acyl carrier protein (ACP) from higher plants in order to develop an assay for plant ACP, and to prepare labeled acyl-ACP of plant origin. It was found that the E. coli enzyme was able to acylate ACP from spinach, soybean, avocado, corn, and several other plants. The acylation was very specific because, in crude extracts of spinach leaves where ACP represented approximately 0.1% of the total soluble protein, ACP was shown to be the only protein acylated. In contrast to other E. coli enzymes that display 2- to 10-fold lower rates with plant versus bacterial ACP, the kinetic constants (Km and Vmax) for acyl-ACP synthetase were found to be essentially identical for spinach and E. coli ACP when acylated with palmitic acid. Palmitic, myristic, lauric, stearic, and oleic acid could all be esterified to both spinach and E. coli ACP with similar specificity. Procedures are described that allow the assay of ACP in plant extracts at the nanogram level.

Acylation↗
PubMed1998

Palmitoylation of proteolipid protein from rat brain myelin using endogenously generated 18O-fatty acids.

Proteolipid protein (PLP), the major protein of central nervous system myelin, contains covalently bound fatty acids, predominantly palmitic acid. This study adapts a stable isotope technique (Kuwae, T., Schmid, P. C., Johnson, S. B., and Schmid, H. O. (1990) J. Biol. Chem. 265, 5002-5007) to quantitatively determine the minimal proportion of PLP molecules which undergo palmitoylation. In these experiments, brain white matter slices from 20-day-old rats were incubated for up to 6 h in a physiological buffer containing 50% H218O. The uptake of 18O into the carbonyl groups of fatty acids derived from PLP, phospholipids, and the free fatty acid pool was measured by gas-liquid chromatography/mass spectrometry of the respective methyl esters. Palmitic acid derived from PLP acquired increasing amounts of 18O, ending with 2.9% 18O enrichment after 6 h of incubation. 18O incorporation into myelin free palmitic acid also increased over the course of the incubation (67.2% 18O enrichment). After correcting for the specific activity of the 18O-enriched free palmitic acid pool, 7.6% of the PLP molecules were found to acquire palmitic acid in 6 h. This value is not only too large to be the result of the palmitoylation of newly synthesized PLP molecules, it was also unchanged upon the inhibition of protein synthesis with cycloheximide. 18O enrichment in less actively myelinating 60-day-old rats was significantly reduced. In conclusion, our experiments suggest that a substantial proportion of PLP molecules acquire palmitic acid via an acylation/deacylation cycle and that this profile changes during development.

Animals↗
PubMed1992

Volume-regulatory Cl- channel currents in cultured human epithelial cells.

1. During osmotic swelling, cultured human small intestinal epithelial cells (Intestine 407) exhibited activation of large Cl- currents under the patch-clamp whole-cell configuration. The volume-sensitive Cl- conductance was independent of intracellular Ca2+ and cyclic AMP. 2. The anion permeability sequence of the current was SCN- > I- > Br- > Cl- > F- > gluconate-, corresponding to Eisenman's sequence I. 3. Cl- currents were instantaneously activated by command pulses in a range of -120 to +45 mV. At potentials more positive than +50 mV the current showed a time-dependent inactivation. This inactivation was accelerated by increased depolarization. The instantaneous current-voltage relationship rectified in the outward direction. 4. A stilbene-derivative Cl- channel blocker, 4-acetamido-4'-isothiocyanostilbene (SITS), inhibited the Cl- current at micromolar concentrations. SITS facilitated inactivation at positive potentials. Outward currents were more prominently suppressed by SITS than inward currents. The concentrations required for 50% inhibition (IC50) of outward and inward currents were 1.5 and 6 microM, respectively. The outward and inward currents were equally inhibited by a carboxylate analogue Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or diphenylamine-2-carboxylate (DPC) at higher doses (IC50 = 25 for NPPB or 350 microM for DPC). Inactivation kinetics at large depolarizations was not affected by NPPB or DPC. 5. The Cl- current was blocked by an unsaturated fatty acid, arachidonic acid (IC50 = 8 microM). Arachidonic acid was still effective in the presence of inhibitors of lipoxygenase (nordihydroguaiaretic acid, 10 microM), cyclo-oxygenase (indomethacin, 10 microM) and protein kinase C (polymyxin B, 30 microM). The Cl- current was also sensitive to another cis unsaturated fatty acid, oleic acid, which is not a substrate for oxygenases. A trans isomer of oleate, elaidic acid, and a saturated fatty acid, palmitic acid, were ineffective. 6. Single Intestine 407 cells exposed to a hypotonic solution showed a regulatory volume decrease after initial osmotic swelling. The volume regulation was abolished by SITS, NPPB, arachidonate and oleate, but not by elaidate and palmitate. 7. It is concluded that outwardly rectifying Cl- channels, which are sensitive to arachidonic acid, are activated upon osmotic swelling and involved in the subsequent cell volume regulation.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1992

Metabolic labeling of glycosylphosphatidylinositol-anchor of heparan sulfate proteoglycans in rat ovarian granulosa cells.

The glycosylphosphatidylinositol (GPI)-anchor of the plasma membrane-associated heparan sulfate (HS) proteoglycan was metabolically radiolabeled with [3H]myristic acid, [3H]palmitic acid, [3H]inositol, [3H]ethanolamine, or [32P]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [3H]myristic acid or [3H]palmitic acid were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100 and the proteoglycans were purified by ion exchange chromatography after extensive delipidation. Specific incorporation of 3H into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of 3H activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of 3H in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that 3H label was the result of the metabolic utilization of catabolic products of the 3H-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [3H]inositol, [3H]ethanolamine, or [32P]phosphate specifically released radiolabeled cell surface-associated HS proteoglycans indicating the presence of GPI-anchor in these proteoglycans. GPI-anchored HS proteoglycans accounted for 20-30% of the total cell surface-associated HS proteoglycans and virtually all of them were removed by PI-PLC. These results further substantiate the presence of GPI-anchored heparan sulfate proteoglycan in ovarian granulosa cells and its cell surface localization.

Animals↗
PubMed1994

Preparation of anti-HIV-low-density lipoprotein complexes for delivery of anti-HIV drugs via the low-density lipoprotein pathways.

Lipophilic prodrugs of 3'-azido-3'-deoxythymidine (AZT) and of 2',3'-didehydro-3'-deoxythymidine (D4T) have been synthesized. 3 beta-(2'-carboxymethoxy)-cholest-5-ene acid, palmitic acid, linolenic acid, linoleic acid, and cholanic acid have been covalently bound to AZT and D4T. In some experiments the fluorescent molecule NBD was simultaneously linked. These prodrugs were incorporated into LDL or acetylated LDL. The best incorporation was obtained with drugs presenting a steroid moiety (cholesterol derivative or cholanic acid) in their structure. The incorporation of prodrugs into LDL was estimated as approximately 200 molecules of prodrug per LDL particle. Cytofluorimetric studies clearly show that the NBD-steroid LDL or NBD-steroid acetylated LDL are bound and then internalized by the B-E receptor (U937) or the scavenger receptor (mouse peritoneal macrophage), respectively. The antiretroviral activity of palmitate-D4T, cholanic-AZT, and cholanic-AZT-LDL complex was similar to the activity of free D4T and free AZT, respectively. Development of lipid nucleoside-LDL complexes to attach specifically to cells involved in HIV infection might have a direct clinical relevance.

Animals↗