Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides

The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1'-8' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2'-13' or 3'-18', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1'-12' and longer whereas rho 1C5 required peptide 1'-18'. Peptide 3'-18' was as effective as 1'-18' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the seven amino acid C-terminus from rhodopsin and further cleavage to F1 (Mr 25 000) and F2 (Mr 12 000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interrelations between ureogenesis and gluconeogenesis in isolated hepatocytes. The role of antion transport and the competition for energy.

1. Glucose synthesis from lactate plus pyruvate and from lactate plus alanine was measured in the presence or absence of 1mM-oleate or 2mM-octanoate at low (2mM) or high (8mM) concentrations of NH4Cl. 2. Both fatty acids alone or with 2mM-NH4Cl doubled glucose production from lactate plus pyruvate. Glucose synthesis from lactate plus alanine, in the presence of oleate, was decreased 16% by 2mM-NH4Cl. 3. In the presence of fatty acids, 8mM-NH4Cl decreased gluconeogenesis by 60-65% from both lactate plus pyruvate and lactate plus alanine. This inhibition was correlated with a high accumulation of aspartate and a drastic decrease in 2-oxoglutarate and malate in the cells. 4. In the presence of 2mM- or 8 mM-NH4Cl, oleate and glucogenic precursors, the addition of 2.5mM-ornithine stimulated urea synthesis. 5. This was paralleled by a decrease of 16% in glucose synthesis from lactate plus pyruvate in the presence of 2mM-NH4Cl and had no effect at 8mM-NH4Cl. In the system producing glucose from lactate plus alanine, ornithine completely reversed the inhibition caused by 2mM-NH4Cl and only partly that by 8mM-NH4Cl. 6. Gluconeogenesis from pyruvate was also inhibited by 2mM-NH4Cl in the presence of oleate or ethanol. This way due to the decrease of malate, which is the C4 precursor of glucose in this system. 7. The limitation of gluconeogenesis by 2-oxoglutarate and malate concentrations in the liver cell and the competition for energy between glucose and urea synthesis is discussed.     

Neighboring group participation. Part 16. Stereoselective synthesis and receptor-binding examination of the four stereoisomers of 16-bromomethyl-3,17-estradiols

The four possible isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a-4a) with proven configurations were converted into the corresponding 3-benzyloxy-16-bromomethylestra-1,3,5(10)-triene-3,17-diols (5e-8e). Depending on the reaction conditions the cis isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a and 2a) were transformed into 3-benzyloxy-16-bromomethylestra-1,3,5(10)-trien-17-yl acetate (5b and 6b) or 16-bromomethyl-3-hydroxyestra-1,3,5(10)-trien-17-yl acetate (5c and 6c) on treatment with HBr and acetic acid. The mechanism of the process can be interpreted as involving front-side neighboring group participation. Under similar experimental conditions, the trans isomers (3a and 4a) yielded only 3-benzyloxy-16-acetoxymethylestra-1,3,5(10)-trien-17-yl acetates (3b and 4b) or 16-acetoxymethylestra-1,3,5(10)-triene-3,17-diyl diacetates (3d and 4d). Both the cis (1a and 2a) and the trans (3a, and 4a) isomers were transformed into 16-bromomethylestra-1,3,5(10)-trien-17-ol (5a-8a) by the Appel reaction on treatment with CBr4/Ph3P. Debenzylation of 5a-8a was carried out with HBr and acetic acid to yield 5e-8e. The debenzylation process in the presence of acetic anhydride produces the diacetates 5d-8d. The structures of the compounds were determined by means of MS, 1H NMR and 13C NMR spectroscopic methods. Compounds 5c-8c and 5e-8e were tested in a radioligand-binding assay. Except for the affinity of 7e for the estrogen receptor (Ki=2.55 nM), the affinities of the eight compounds (5c-8c and 5e-8e) for the estrogen, androgen and progesterone receptors are low (Ki > 0.55, 0.52 and 0.21 microM, respectively).     

Studies on the glycosphingolipids of the starfish, Asterina pectinifera. II. Isolation and characterization of a novel ganglioside with an internal sialic acid residue.

1. Three gangliosides, provisionally named Gangliosides 1, 2, and 3, were obtained from the lipid extract of the starfish, Asterina pectinifera by silicic acid, DEAE-Sephadex, and Iatrobeads column chromatography. The most abundant, Ganglioside 3 (37.7 microgram/g wet weight of starfish) was isolated in the pure state and its chemical structure was studied. 2. The sugar composition of Ganglioside 3 consisted of arabinose, glucose, galactose, and sialic acid (as N-glycolylneuraminic acid) in a molar ratio of 1:1:3:1. Three sialic acid-containing oligosaccharide fragments were isolated from partial acid hydrolysates of the ganglioside by Dowex 1 X 8 (acetate form) column chromatography and preparative paper chromatography, and identified as Gal leads to NeuGc, Gal (1 leads to 4)[Gal(1 leads to 8)]NeuGc and Ara-(1 leads to 6)Gal(1 leads to 4)[Gal(1 leads to 8)]NeuGc. 3. The structure of Ganglioside 3 was postulated to be: Araf,p(1 leads to 6)Galpbeta(1 leads to 4)[Galpbeta(1 leads to 8)]NeuGc(2 leads to 3)Galpbeta(1 leads to 4)Glcpbeta(1 leads to 1)-ceramide. This is a unique structure with the sialic acid residue internally located in the sugar chain. 4. The ganglioside contained saturated 2-hydroxy fatty acids ranging in length from C16 to C24, among which C22, C23, and C24 acids were predominant. The long-chain bases consisted exclusively of C16, C17, and C18 phytosphingosines of iso and anteiso types.     

N,N'-dicyclohexylcarbodiimide-binding proteolipid of the vacuolar H+-ATPase from oat roots

The inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was used to probe the structure and function of the vacuolar H+-translocating ATPase from oat roots (Avena sativa var. Lang). The second-order rate constant for DCCD inhibition was inversely related to the concentration of membrane, indicating that DCCD reached the inhibitory site by concentrating in the hydrophobic environment. [14C]DCCD preferentially labeled a 16-kDa polypeptide of tonoplast vesicles, and the amount of [14C]DCCD bound to the 16-kDa peptide was directly proportional to inhibition of ATPase activity. A 16-kDa polypeptide had previously been shown to be part of the purified tonoplast ATPase. As predicted from the observed noncooperative inhibition, binding studies showed that 1 mol of DCCD was bound per mol of ATPase when the enzyme was completely inactivated. The DCCD-binding 16-kDa polypeptide was purified 12-fold by chloroform/methanol extraction. This protein was thus classified as a proteolipid, and its identity as part of the ATPase was confirmed by positive reaction with the antibody to the purified ATPase on immunoblots. From the purification studies, we estimated that the 16-kDa subunit was present in multiple (4-8) copies/holoenzyme. The purification of the proteolipid is a first step towards testing its proposed role in H+ translocation.     

Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. Studies with chemical modifiers of amino acid residues

Possible involvement of polypeptides of b-c1 complex of beef-heart mitochondria in its redox and protonmotive activity has been investigated, by means of chemical modification of amino acid residues in the soluble as well as in the phospholipid-reconstituted b-c1 complex. Treatment of the enzyme with tetranitromethane (C(NO2)4) or with ethoxyformic anhydride (EFA), that modify reversibly tyrosyl and hystidyl residues respectively, resulted in a marked inhibition of electron transport from reduced quinols to cytochrome c. This was accompanied, in b-c1 reconstituted into phospholipid vesicles, by a parallel inhibition of respiratory-linked proton translocation; the H+/e- stoichiometry remained unchanged. Treatment of b-c1 complex with DCCD, that specifically modifies carboxylic groups of glutammic or aspartic residues caused a marked depression of proton translocation in b-c1 vesicles, under conditions where the rate of electron flow in the coupled state, was enhanced. As a consequence the H+/e- stoichiometry was lowered. SDS gel electrophoresis and [14C]DCCD-labelling of the polypeptides of the b-c1 complex showed a major binding of 14C-DCCD to the 8-kDa subunit of the complex and possible cross-linking, induced by DCCD treatment, of polypeptide(s) in the 8-kDa band and the 12-kDa band, with the Fe-s protein of the complex, with the appearance of a new polypeptide band with an apparent molecular mass of about 40 kDa. Involvement of polypeptides of low molecular mass, for which no functional role was so far described, and possibly of the Fe-S protein in the redox-linked proton translocation in b-c1 complex is suggested.     

Inhibitory activity of spices and essential oils on psychrotrophic bacteria

This study was designed to evaluate "in vitro" the inhibitory effects of spices and essential oils on the growth of psycrotrophic food-borne bacteria: Aeromonas hydrophila, Listeria monocytogenes and Yersinia enterocolitica. The sensitivity to nine spices and their oils (chilli, cinnamon, cloves, ginger, nutmeg, oregano, rosemary, sage, thyme) was studied. Antibacterial activity was evaluated on liquid and solid medium. Spices: 1% concentration of each spice was added separately to Triptic Soy Broth and then inoculated to contain 10(8)/ml organism and held to 4 degrees C for 7 days. Populations of test organism were determined on Triptic Soy Agar. Oils: Inhibition of growth was tested by using the paper disc agar diffusion method (at 35, 20 and 4 degrees C) and measuring their inhibition zone. MIC was determined by the broth microdilution method. Some culinary spices produce antibacterial activity: inhibition of growth ranged from complete (cinnamon and cloves against A. hydrophila) to no inhibition. Antibacterial inhibition zone ranged from 8 mm to 45 mm: thyme essential oil showed the greatest inhibition against A. hydrophila.     

Enzymatic properties of purified murine fatty acid transport protein 4 and analysis of acyl-CoA synthetase activities in tissues from FATP4 null mice

Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using alpha-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V(max)/K(m) (C16:0)/V(max)/K(m) (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.     

Synthesis of single- and double-13C-labeled cholesterol oleate

Cholesterol oleate with the 13C-label in oleic acid at the carbonyl and/or in the sterol ring at position 4 was synthesized by two methods: (1) cholesterol was condensed with oleic anhydride, prepared from [1-13C] oleic acid, in the presence of dimethylaminopyridine (DMAP) in anhydrous chloroform at room temperature for 4--5 h; (2) cholesterol or 13C-enriched cholesterol at position 4 were reacted with 90% [1-13C]-oleic acid in the presence of dicyclohexylcarbodiimide (DCC) and DMAP at room temperature in anhydrous chloroform for 1.25 h. The single-13C and double-13C-labeled cholesterol oleate were obtained in 90% yields after purification by silicic acid column chromatography. Their purity was assessed by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and 13C-NMR spectroscopy. Tritium-labeled cholesterol oleate was also synthesized by method 1 using the fatty acid anhydride.     

Modulation of phagocytosis-associated respiratory burst by human cystatin C: role of the N-terminal tetrapeptide Lys-Pro-Pro-Arg

Cystatin C, a cysteine protease inhibitor, has recently been suggested to be a potent regulator in inflammatory processes. Human cystatin C was isolated from the urine of one patient suffering from tubular disorders and was tested for its effects on two functions of human polymorphonuclear neutrophils (PMN): O2- release and phagocytosis. Slow-form or (des 1-4) cystatin C and fast-form or (des 1-8) cystatin C differed by the presence in (des 1-4) cystatin C only of the N-terminal tetrapeptide Lys-Pro-Pro-Arg. Whereas (des 1-8) cystatin C did not seem to interfere with PMN functions at physiological concentrations, (des 1-4) cystatin C induced an inhibition of PMN phagocytosis-associated respiratory burst in response to opsonized zymosan particles. The inhibition may be attributed to the tetrapeptide Lys-Pro-Pro-Arg which has been synthesized and shown to have the same inhibitor effects, at concentrations similar to those required for (des 1-4) cystatin C. These results support a potential role for cystatin C as a modulator during inflammation.     

The role of Delta(9)-desaturase in the production of cis-9, trans-11 CLA

Biomedical studies with animal models have demonstrated that cis-9, trans-11 conjugated linoleic acid (CLA), the predominant isomer found in milk fat from dairy cows, has anticarcinogenic effects. We recently demonstrated endogenous synthesis of cis-9, trans-11 CLA from ruminally derived trans-11 C18:1 by Delta(9)-desaturase in lactating dairy cows. The present study further examined endogenous synthesis of cis-9, trans-11 CLA and quantified its importance by increasing substrate supply using partially hydrogenated vegetable oil (PHVO) as a source of trans-11 C18:1 and blocking endogenous synthesis using sterculic oil (SO) as a source of cyclopropene fatty acids which specifically inhibit Delta(9)-desaturase. Four cows were abomasally infused with 1) control, 2) PHVO, 3) SO, and 4) PHVO+SO in a 4 x 4 Latin square design. With infusion of PHVO, cis-9, trans-11 CLA was increased by 17% in milk fat. Consistent with inhibition of desaturase, SO treatments increased milk fat ratios for the fatty acid pairs effected by Delta(9)-desaturase, C14:0/cis-9 C14:1, C16:0/cis-9 C16:1, and C18:0/cis-9 C18:1. The role of endogenous synthesis of CLA was evident from the 60-65% reduction in cis-9, trans-11 CLA which occurred in milk fat with SO treatments. cis-9 C14:1 originates from desaturation of C14:0 by Delta(9)-desaturase and can be used to estimate the extent of SO inhibition of Delta(9)-desaturase. When this correction factor was applied, endogenous synthesis was estimated to account for 78% of the total cis-9, trans-11 CLA in milk fat. Thus, endogenous synthesis was the major source of cis-9, trans-11 CLA in milk fat of lactating cows.     

Incorporation and remodeling of extracellular phosphatidylcholine with short acyl residues in Saccharomyces cerevisiae

The pem1/cho2 pem2/opi3 double mutant of Saccharomyces cerevisiae, which is auxotrophic for choline because of the deficiency in methylation activities of phosphatidylethanolamine, grew in the presence of 0.1 mM dioctanoyl-phosphatidylcholine (diC(8)PC). Analysis of the metabolism of methyl-(13)C-labeled diC(8)PC ((methyl-(13)C)(3)-diC(8)PC) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that it was rapidly converted to (methyl-(13)C)(3)-PCs containing C16 or C18 acyl chains. (Methyl-(13)C)(3)-8:0-lyso-PC, (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC, which are the probable intermediate molecular species of acyl chain remodeling, appeared immediately after 5 min of pulse-labeling and decreased during the subsequent chase period. These results indicate that diC(8)PC was taken up by the pem1 pem2 double mutant and that the acyl chains of diC(8)PC were exchanged with longer yeast fatty acids. The temporary appearance of (methyl-(13)C)(3)-8:0-lyso-PC suggests that the remodeling reaction may consist of deacylation and reacylation by phospholipase activities and acyltransferase activities, respectively. The detailed analyses of the structures of (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC by MS/MS and MS(3) strongly suggest that most (methyl-(13)C)(3)-8:0-16:0-PCs have a C16:0 acyl chain at sn-1 position, whereas (methyl-(13)C)(3)-8:0-16:1-PCs have a C16:1 acyl chain at either sn-1 or sn-2 position in a similar frequency, implying that the initial C16:0 acyl chain substitution prefers the sn-1 position; however, the C16:1 acyl chain substitution starts at both sn-1 and sn-2 positions. The current study provides a pivotal insight into the acyl chain remodeling of phospholipids in yeast.     

Surface properties of sulfur- and ether-linked phosphonolipids with and without purified hydrophobic lung surfactant proteins

Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO(2)-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8 and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins (SP)-B and/or SP-C. Synthetic C16:0 phosphonolipids all had improved adsorption and film respreading compared to dipalmitoyl phosphatidylcholine, and SO(2)-lipid and DEPN-8 reached maximum surface pressures of 72mN/m (minimum surface tensions of <1mN/m) in compressed films on the Wilhelmy balance (23 degrees C). Dispersions of DEPN-8 (0.5mg/ml) and SO(2)-lipid (2.5mg/ml) also reached minimum surface tensions of <1mN/m on a pulsating bubble surfactometer (37 degrees C, 20cycles/min, 50% area compression). Synthetic lung surfactants containing DEPN-8 or SO(2)-lipid+0.75% SP-B+0.75% SP-C had dynamic surface activity on the bubble equal to that of calf lung surfactant extract (CLSE). Surfactants containing DEPN-8 or SO(2)-lipid plus 1.5% SP-B also had very high surface activity, but less than when both apoproteins were present together. Adding 10wt.% of UnDEPN-8 to synthetic lung surfactants did not improve dynamic surface activity. Surfactants containing DEPN-8 or SO(2)-lipid plus 0.75% SP-B/0.75% SP-C were chemically and biophysically resistant to phospholipase A(2) (PLA(2)), while CLSE was severely inhibited by PLA(2). The high activity and inhibition resistance of synthetic surfactants containing DEPN-8 or SO(2)-lipid plus SP-B/SP-C are promising for future applications in treating surfactant dysfunction in inflammatory lung injury.     

Synthesis,biological evaluation and molecular docking study of 1,2,3-1H-triazoles having 4H-pyrano[2,3-d]pyrimidine as potential Mycobacterium tuberculosis protein tyrosine phosphatase B inhibitors

Some heterocycles, namely 2-amino-4H-pyran-3-carbonitriles, were synthesized in a three-component reaction from substituted benzaldehydes, malononitrile, and ethyl acetoacetate. These heterocycles have been converted subsequently into 4H-pyrano[2,3-d]pyrimidine ring by ring-closing reaction with acetic anhydride in the presence of the concentrated sulfuric acid as catalyst. The successive alkylation reaction of lactam NH bond on pyrimidine-4-one ring was carried out using propargylic bromide in dry acetone in the presence of anhydrous potassium carbonate. The click chemistry of 3-propargyl-4H-pyrano[2,3-d]pyrimidine compounds has been accomplished by reaction with tetra-O-acetyl-α-d-glucopyranosyl azide using the metal-organic framework Cu@MOF-5 as a catalyst in absolute ethanol. All the synthesized 1H-1,2,3-triazoles 8a–y were screened for their in vitro Mycobacterium tuberculosis protein tyrosine phosphatase B (MtbPtpB) inhibition. Kinetic studies of the most active compounds 8v, 8x, and 8y showed their competitive inhibition toward the MtbPtpB enzyme. The detailed structure-activity relationship (SAR) in vitro and in silico studies suggested that the interaction of Arg63 amino acids with anion type of para-hydroxyl group via a salt bridge of iminium cation was essential for strong inhibitory activity against MtbPtpB.     

C(4)-alkyl substituted furanyl cyclobutenediones as potent, orally bioavailable CXCR2 and CXCR1 receptor antagonists

A novel series of cyclobutenedione centered C(4)-alkyl substituted furanyl analogs was developed as potent CXCR2 and CXCR1 antagonists. Compound 16 exhibits potent inhibitory activities against IL-8 binding to the receptors (CXCR2 Ki=1 nM, IC(50)=1.3 nM; CXCR1 Ki=3 nM, IC(50)=7.3 nM), and demonstrates potent inhibition against both Gro-alpha and IL-8 induced hPMN migration (chemotaxis: CXCR2 IC(50)=0.5 nM, CXCR1 IC(50)=37 nM). In addition, 16 has shown good oral pharmacokinetic profiles in rat, mouse, monkey, and dog.     

The effect of cationic surface-active agents on the Escherichia coli ATPase

The authors studied the effect of cationic surfactants (CS), such as alkyl(C8H17-C18H37)dimethylbenzylammonium (I), alkyl(C8H17-C16H33)benzyltrimethylammonium (II), alkyl(C8H17-C16H33)di-beta-hydroxyethylbenzylammonium (III) chlorides and chlorhydrate of glycine decyl ester (IV) on the ATPase activity of E. coli 1257 cell, spheroplasts, and isolated membranes. Changes in the ATPase activity of the E. coli cells and spheroplasts were found to depends on the concentration and the structure of the cationic surfactants. The removal of the cell wall increased the destroying effect of CS on the cytoplasmic membranes and enhanced the ATPase inhibition. The compounds with 16 and 18 carbon atom radical had the highest inhibitory effect. The action of cationic surfactants on the membrane is accompanied by changes in the protein and phospholipid composition and by significant solubilization of ATPase with pronounced inactivation of the enzyme. The kinetics of inhibition of E. coli membrane ATPase was studied to the presence of the homological series I and IV. The cationic surfactants under study inhibited the ATP hydrolysis catalysed by E. coli ATPase by a mixed type mechanism. Ki = 58.21.10(-4) M for IC10H21; 10.67.10(-4) M for IC12H25; 0.58.10(-4) M for IC16H33; 0.16.10(-4) M for IC18H37, and 5.93.10(-4) M for IV.     

Fatty acids of pulmonary surfactant phosphatidylcholine from fetal rabbit lung tissue in culture. Biosynthesis of n-10 monoenoic fatty acids

We have previously reported that fetal rabbit lung tissue in organ culture produces a lamellar body material (pulmonary surfactant) with a lower percentage of disaturated phosphatidylcholine than is typically found in rabbit lung in vivo (Longmuir, K.J., C. Resele-Tiden, and L. Sykes. 1985. Biochim. Biophys. Acta. 833: 135-143). This investigation was conducted to identify all fatty acids present in the lamellar body phosphatidylcholine, and to determine whether the low level of disaturated phosphatidylcholine is due to excessive unsaturated fatty acid at position sn-1, sn-2, or both. Fetal rabbit lung tissue, 23 days gestation, was maintained in culture for 7 days in defined (serum-free) medium. Phospholipids were labeled in culture with [1-14C]acetate or [U-14C]glycerol (to follow de novo fatty acid biosynthesis), or with [1-14C]palmitic acid (to follow incorporation of exogenously supplied fatty acid). Radiolabeled fatty acid methyl esters obtained from lamellar body phosphatidylcholine were first separated by reverse-phase thin-layer chromatography (TLC) into two fractions of 1) 14:0 + 16:1 and 2) 16:0 + 18:1. Complete separation of the individual saturated and monoenoic fatty acids was achieved by silver nitrate TLC of the two fractions. Monoenoic fatty acid double bond position was determined by permanganate-periodate oxidation followed by HPLC of the carboxylic acid phenacyl esters. Lamellar body phosphatidylcholine contained four monoenoic fatty acids: 1) palmitoleic acid, 16:1 cis-9; 2) oleic acid, 18:1 cis-9; 3) cis-vaccenic acid, 18:1 cis-11; and 4) 6-hexadecenoic acid, 16:1 cis-6. In addition, 8-octadecenoic acid, 18:1 cis-8, was found in the fatty acids of the tissue homogenate. The abnormally low disaturated phosphatidylcholine content in lamellar body material was the result of abnormally high levels of monoenoic fatty acid (principally 16:1 cis-9) found at position sn-2. Position sn-1 contained normal levels of saturated fatty acid. The biosynthesis of the unusual n-10 fatty acids was observed from the start of culture throughout the entire 7-day culture period, and was observed in incubations of tissue slices of day 23 fetal rabbit lung. This is the first report of the biosynthesis of n-10 fatty acids (16:1 cis-6 and 18:1 cis-8) in a mammalian tissue other than skin, where these fatty acids are found in the secretory product (sebum) of sebaceous glands.     

Effect of sugars in the preservation solution on liver storage in rats.

We have performed 128 rat liver transplants in order to examine the effect of sugars in preservation solutions on cold storage of rat livers. Glucose (Mw. 180), sucrose (Mw. 348), and raffinose (Mw. 594) were tested. Rat livers were preserved at 4 degrees C for 12, 16, 18, and 24 h in standard Eurocollins solution (EC solution) (solution A) or in one of three modified EC solutions in which 194 mM/liter glucose in standard EC solution was replaced by 140 mM/liter of glucose (solution B), sucrose (solution C), or raffinose (solution D). The osmolarity of the modified solutions (solution B-D) was 320 mOsm/liter. Using standard EC solution (solution A), the 1-week survival rate of rats receiving livers preserved for 12, 16, 18, or 24 h was 6/8, 4/8, 1/8, and 0/4, respectively. With solution B, in which 194 mM/liter glucose was replaced by 140 mM/liter glucose, 1 week survivors following transplantation of livers preserved for 12, 16, 18 or 24 h were 4/8, 3/8, 2/8 and 0/4, respectively. Solution C, which was identical to solution A except for the replacement of 194 mM/liter glucose by 140 mM/liter sucrose, gave the following 1-week survival rates: 5/8 for 12 h, 5/8 for 16 h, 2/8 for 18 h, and 0/4 for 24 hours preservation, respectively. Using solution D, which differed from A in the replacement of glucose by 140 mM/liter raffinose, the 1-week survival rates of rats grafted with livers preserved for 12, 16, 18, and 24 h were 6/8, 5/8, 3/8 and 0/4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

How alfalfa root hairs discriminate between Nod factors and oligochitin elicitors

Using ion-selective microelectrodes, the problem of how signals coming from symbiotic partners or from potential microbial intruders are distinguished was investigated on root hairs of alfalfa (Medicago sativa). The Nod factor, NodRm-IV(C16:2,S), was used to trigger the symbiotic signal and (GlcNAc)(8) was selected from (GlcNAc)(4-8), to elicit defense-related reactions. To both compounds, root hairs responded with initial transient depolarizations and alkalinizations, which were followed by a hyperpolarization and external acidification in the presence of (GlcNAc)(8). We propose that alfalfa recognizes tetrameric Nod factors and N-acetylchitooligosaccharides (n = 4-8) with separate perception sites: (a) (GlcNAc)(4) and (GlcNAc)(6) reduced the depolarization response to (GlcNAc)(8), but not to NodRm-IV(C16:2, S); and (b) depolarization and external alkalization were enhanced when NodRm-IV(C16:2,S) and (GlcNAc)(8) were added jointly without preincubation. We suggest further that changes in cytosolic pH and Ca(2+) are key events in the transduction, as well as in the discrimination of signals leading to symbiotic responses or defense-related reactions. To (GlcNAc)(8), cells responded with a cytosolic acidification, and they responded to NodRm-IV(C16:2,S) with a sustained alkalinization. When both agents were added jointly, the cytosol first alkalized and then acidified. (GlcNAc)(8) and NodRm-IV(C16:2,S) transiently increased cytosolic Ca(2+) activity, whereby the response to (GlcNAc)(8) exceeded the one to NodRm-IV(C16:2,S) by at least a factor of two.     

On the singular, dual, and multiple positional specificity of manganese lipoxygenase and its G316A mutant

Abstract manganese lipoxygenase (Mn-LO) oxygenates 18:3n-3 and 18:2n-6 to bis-allylic 11S-hydroperoxy fatty acids, which are converted to 13R-hydroperoxy fatty acids. Other unsaturated C(16)-C(22) fatty acids, except 17:3n-3, are poor substrates, possibly because of ineffective enzyme activation (Mn(II)-->Mn(III)) by the produced hydroperoxides. Our aim was to determine whether unsaturated C(16)-C(22) fatty acids were oxidized by Mn(III)-LO. Mn(III)-LO oxidized C(16), C(19), C(20), and C(22) n-3 and n-6 fatty acids. The carbon chain length influenced the position of hydrogen abstraction (n-8, n-5) and oxygen insertion at the terminal or the penultimate 1Z,4Z-pentadienes. Dilinoleoyl-glycerophosphatidylcholine was oxidized by Mn-LO, in agreement with a "tail-first" model. 16:3n-3 was oxidized at the bis-allylic n-5 carbon and at positions n-3, n-7, and n-6. Long fatty acids, 19:3n-3, 20:3n-3, 20:4n-6, 22:5n-3, and 22:5n-6, were oxidized mainly at the n-6 and the bis-allylic n-8 positions (in ratios of approximately 3:2). The bis-allylic hydroperoxides accumulated with one exception, 13-hydroperoxyeicosatetraenoic acid (13-HPETE). Mn(III)-LO oxidized 20:4n-6 to 15R-HPETE ( approximately 60%) and 13-HPETE ( approximately 37%) and converted 13-HPETE to 15R-HPETE. Mn(III)-LO G316A oxygenated mainly 16:3n-3 at positions n-7 and n-6, 19:3n-3 at n-10, n-8, and n-6, and 20:3n-3 at n-10 and n-8. We conclude that Mn-LO likely binds fatty acids tail-first and oxygenates many C(16), C(18), C(20), and C(22) fatty acids to significant amounts of bis-allylic hydroperoxides.