Variant specific glycoproteins of Trypanosoma equiperdum: cross reacting determinants and chemical studies

Two variant specific surface antigens (VSSA) (BoTat-1 and BoTat-28) were purified from clones of $\text{Trypanosoma equiperdum}$ and tested for immunological cross reactivity. Competitive radioimmunoassay revealed the presence of cross reacting determinants. Limited tryptic cleavage of BoTat-28 resulted in the purification of two fragments F₁ and F₂ with respectively 38,000 and 28,000 apparent molecular weight. F₁ represents a large N-terminal fragment containing 30 % of the total sugar content. F₂ is characterized by an N-terminal amino acid sequence different from that of the native glycoprotein and contains all the carbohydrate. Inhibition of the heterologous precipitation reaction was only achieved with F₂ and reaches 100 % as with the native glycoproteins. These results demonstrate the presence of cross reacting determinants located in the C-terminal part of the molecule.     

Temperature adaptation of biological membranes. The effects of acclimation temperature on the unsaturation of the main neutral and charged phospholipids in mitochondrial membranes of the carp (cyprinus carpio L.)

The phospholipid composition, fatty acid pattern and cholesterol content are studied in mitochondria of red lateral muscle of carp acclimated to high and low environmental temperatures.The results of the experiments are: mitochondria from cold-acclimated carp contain higher proportions of ethanolamine phosphatides than mitochondria from warm-acclimated fish, the opposite is true for the choline phosphatides. Thus, at constant pH, the membrane phospholipids are slightly more negatively charged at low acclimation temperature. The total plasmalogen content is reduced in the cold; this reduction is caused by a decrease in the proportion of the choline plasmalogens. The ethanolamine phosphoglycerides contain approx. 20% of the alk-1-enyl acyl type, irrespective of the acclimation temperature. There is no temperature-dependent difference in the low proportion of cholesterol.The fatty acids of total mitochondrial phospholipids are characterized by large amounts of the $\text{n-3}\text{and}\text{n-6}$ families. The ratio of unsaturated to saturated fatty acids and the unsaturation index are remarkably higher than those reported for comparable mammalian phospholipids. Cold acclimation of carp does not significantly increase the unsaturation of total phospholipids. A fatty acid analysis of the main isolated phospholipids, however, shows that cold acclimation considerably increases unsaturation of the neutral phosphatidylcholine, whereas it dramatically decreases unsaturation of the negatively charged cardiolipin. It is suggested that the observed fatty acid substitution in phosphatidylcholine indicates a temperature-induced fluidity adaptation within the mitochondrial lipid bilayer, whereas the inverse acclimation pattern of cardiolipin provides a suitable lipid to accommodate the temperature-dependent modifications in the dynamic surface shape of integral membrane proteins.     

Trypanosoma evansi: Identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain

African trypanosomes are flagellated unicellular parasites which proliferate extracellularly in the mammalian host blood-stream and tissue spaces. They evade the hosts’ antibody-mediated lyses by sequentially changing their variant surface glycoprotein (VSG). VSG tightly coats the entire parasite body, serving as a physical barrier. In Trypanosoma brucei and the closely related species Trypanosoma evansi, Trypanosoma equiperdum, each VSG polypeptide can be divided into N- and C-terminal domains, based on cysteine distribution and sequence homology. N-terminal domain, the basis of antigenic variation, is hypervariable and contains all the exposed epitopes; C-terminal domain is relatively conserved and a full set of four or eight cysteines were generally observed. We cloned two genes from two distinct variants of T. evansi, utilizing RT-PCR with VSG-specific primers. One contained a VSG type A N-terminal domain followed a C-terminal domain lacking cysteine residues. To confirm that this gene is expressed as a functional VSG, the expression and localization of the corresponding gene product were characterized using Western blotting and immunofluorescent staining of living trypanosomes. Expression analysis showed that this protein was highly expressed, variant-specific, and had a ubiquitous cellular surface localization. All these results indicated that it was expressed as a functional VSG. Our finding showed that cysteine residues in VSG C-terminal domain were not essential; the conserved C-terminal domain generally in T. brucei like VSGs would possibly evolve for regulating the VSG expression.     

A NMR study of the ionization of fatty acids,fatty amines and N-acylamino acids incorporated in phosphatidylcholine vesicles

The ionization of fatty acids, fatty amines and $\text{N-}\text{acylamino}$ acids incorporated in phosphatidylcholine single-walled vesicles has been measured. The guest molecules have been specifically enriched with ¹³C and titrated by using NMR spectroscopy. The apparent p$\text{K}{}_{\mathrm{a}}$ of fatty acids in phosphatidylcholine bilayers is 7.2–7.4 and those of fatty amines are approx. 9.5. These p$\text{K}{}_{\mathrm{a}}$ values depend on many different parameters related to the structure of the lipid/ solution interface, to the composition of the aqueous medium and to the localization of the ionizable groups. A special sensitivity to the ionic strength and to the surface charge has been found. A positive surface charge decreases the p$\text{K}{}_{\mathrm{a}}$ value whereas a negative one increases it, the total range of variation being 2.5–3 units. In a qualitative macroscopic interpretation, it is proposed that p$\text{K}{}_{\mathrm{a}}$ is essentially determined by the low polarity of the lipidic matrix.     

Biochimica et Biophysica Acta: Vol. 307 (1973)

The present study evaluates the unsaturated fatty acid requirement in Escherichia coli. A derivative of a double mutant defective both in unsaturated fatty acid biosynthesis and in fatty acid degradation has been selected which grows equally well on anteisopentadecanoate ($\text{12-Me-14:0}$) or $\text{cis-Δ}{}^9\text{-}\text{octadecenoate}$ ($\text{cis-δ}{}^9\text{-18:1}$). When this strain is grown for many generations on $\text{12-Me-14:0}$, there is extensive incorporation of this analogue into the membrane phospholipid and essentially no detectable unsaturated fatty acids residues in any lipid-containing structures of the cell envelope. Secondly, as the maximal growth temperature of E. coli is approached, the minimum content of unsaturated fatty acid required by this strain for growth decreases to a few percent and is associated with the appearance of substantial amounts of $\text{12:0}$ (8%) and $\text{14:0}$ (50%) in the phospholipid. These experiments demonstrate that the cis unsaturated fatty acids of E. coli phospholipids can be replaced by residues which possess no special electronic configuration. Hence, the unsaturated fatty acids do not participate in specific interactions with other membrane components but serve a general role of controlling the packing of paraffin chains in the membrane bilayer.     

Drug-induced changes in lipid composition of E. coli and of mammalian cells in culture: ethanol, pentobarbital, and chlorpromazine.

Both Chinese hamster ovary cells in culture and $\text{E.}$$\text{coli}$ cells change their lipid composition when grown in the presence of ethanol, pentobarbital, and chlorpromazine. The effects of ethanol and the cross-tolerant drug, pentobarbital, are similar. Both cause a shift from 18:0 fatty acid to 16:0 fatty acids in CHO cells and a decrease in the proportion of saturated fatty acids in $\text{E.}$$\text{coli}$. Chlorpromazine, a non-cross-tolerant drug, causes the opposite effect in $\text{E.}$$\text{coli}$, a decrease in the proportion of unsaturated fatty acids. Chlorpromazine has little effect on the fatty acid composition of CHO cells. These changes in lipid composition are proposed as an adaptive response and a part of the mechanism for the development of drug tolerance.     

Coenzyme A: fatty acid synthetase apoenzyme 4'-phosphopantetheine transferase in yeast

A mixture of two pantetheine-free mutant fatty acid synthetases was dissociated and recombined $\text{in}$$\text{vitro}$ to form a hybrid apoenzyme complex. $\text{In}$$\text{vivo}$ the corresponding $\text{Saccharomyces}$$\text{cerevisiae}$$\text{fas}$-mutants exhibit interallelic complementation when crossed with each other and the enzyme synthesized in the resulting diploid contains pantetheine and exhibits overall fatty acid synthetase activity. Accordingly, the hybrid apoenzyme formed $\text{in}$$\text{vitro}$ could be activated to holo-fatty acid synthetase when incubated with coenzyme A and a partially purified yeast cell extract. The enzyme coenzyme A: fatty acid synthetase apoenzyme 4′-phosphopantetheine transferase has thus been identified in yeast. Further studies on the mechanism of fatty acid synthetase holoenzyme formation will now be possible.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of $\text{Escherichia}\text{coli}$ by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of $\text{E.}\text{coli}$, was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD⁺ and NADP⁺, but NADPH served to accelerate the rate of inhibition.     

Lipid metabolism and in vitro production of progesterone and prostaglandin F during induced regression of porcine corpora lutea

The effects of Cloprostenol administration on porcine luteal lipid and arachidonic acid accumulation were examined in relation to luteal $\text{in vitro}$ progesterone and prostaglandin F synthesis in 18 mature gilts at day 12 of the estrous cycle. Basal and net $\text{in vitro}$ release of progesterone from luteal tissue was depressed at 8 hr after treatment whereas net $\text{in vitro}$ release of prostaglandin F was elevated at 8 hr. Inclusion of copper dithiothreitol or reduced glutathione in the incubation media resulted in minor alterations of $\text{in vitro}$ release of progesterone and prostaglandin F and no changes in composition of luteal lipids or fatty acids. Luteal contents of triglyceride had increased by 8 hr after treatment whereas contents of free and esterified cholesterols had increased by 32 hr after Cloprostenol administration. Luteal contents of phospholipid and free fatty acids were not affected by Cloprostenol administration. At 32 hr after treatment, percentages and content of arachidonic acid had increased in luteal cholesterol esters and triglycerides. Although arachidonic acid percentages increased in luteal free fatty acids and phospholipids, calculated arachidonic acid contents did not change following Cloprostenol administration. Induced luteal regression was associated with decreased $\text{in vitro}$ progesterone release, increased $\text{in vitro}$ prostaglandin F release, and accelerated lipid and arachidonic acid accumulation within the corpus luteum. The effects of altered lipid metabolism on release of prostaglandin F could not be defined. However, availability of arachidonic acid did not appear to be rate-limiting in relation to luteal $\text{in vitro}$ prostaglandin F synthesis.     

Effect of E. coli endotoxin on the structure-function of fatty acid synthetase lipoprotein

The lipoprotein structure of the fatty acid synthetase complex from Ceratitis capitata has been used as a model to $\text{val}\text{i}$ date the claim that phospholipids from membranes assume a $\text{signif}\text{i}$ cant role in the cell-endotoxin interactions. The enzyme-complex was exposed to a ¹⁴C-lipopolysaccharide preparation and the $\text{inte}\text{r}$ action was followed by a) circular dichroism spectra, b) enzyme activity and c) gel filtration chromatography. It should be $\text{emph}\text{a}$ sized that the E. coli endotoxin modifies all these properties of the enzyme complex and that a model involving phospholipids and phase transitions has been proposed to account for these $\text{intera}\text{c}$ tions.     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.     

Stereospeific 1H-NMR analysis of trimethylsilyl derivatives of glycerides with a chiral shift reagent

A proton magnetic resonance procedure with tri(3-heptafluorobutyryl-d-camphorato)praseodymium (III) as a chiral shift eagent has been developed to determine the enantimeric purity of monoglycerides 1,2-diglycerides and triglycerides with one mono-unsaturated fatty acid at position $\text{sn-1}$ or $\text{sn-3}$ and two saturated fatty acids at the two other glycerol positions. A model compound, 1-oleoyl-2,3-dipalmitoyl-sn-glycerol, was converted ito the trimethylsilyl either of 2,3-dipalmitoyl-an-glycerol by epoxidation of the double bond, followed by pancreatic hydrolysis and separation and trimethylsilylation of the resulting $\text{sn-1,2}$, and $\text{sn-2,3-}\text{diglycerides}$. This separation becomes feasible by the contribution of the epoxy group to the polarity of the diglyceride. The protons of the trimethysilyl ether group were used for determining the enantiomeric ratio. The addition of a chira shift reagent induces a useful enantiomeric splitting which allows the accurate determination of the ratio of both enantiomers. The trimethylsilyl emers of 1,2-diglycerides are better suited for this purpose than the acetyl compounds. For monoglycetides, the earlier published method with the diaceltates gives a better line separation in ¹H-NMR spectra.     

Metabolism of mevalonic acid to long chain fatty alcohols in an insect

A mixture of long chain fatty alcohols (LCFA) with chain lengths of 24 to 30 carbon atoms, as free and esterified components, have been isolated from the aphid, $\text{Schizaphis}\text{graminum}$. Both radiolabelled mevalonic acid and palmitic acid served as effective precursors in the synthesis of LCFA. This is the first time that the $\text{trans}$-methylglutaconate shunt has been shown to be operational for fatty alcohol synthesis and to occur in a lower-evolved animal.     

Effects of free fatty acids as membrane components on permeability of drugs across bilayer lipid membranes. A mechanism for intestinal absorption of acidic drugs

Studies of the influence of fatty acids, which were the component of intestinal mucosal lipids, on the permeability of several drugs across bilayer lipid membranes generated from egg phosphatidylcholine and intestinal lipid have been pursued. The permeability coefficients of $\text{p-}\text{aminobenzoic}$ acid, salicylic acid and $\text{p-}\text{aminosalicylic}$ acid (anionic-charged drug) increased when fatty acids such as lauric, stearic, oleic, linoleic and linolenic acid were incorporated into the bilayer lipid membranes generated from phosphatidylcholine. In the presence of methyl linoleate and oleyl alcohol, no enhancing effect on $\text{p-}\text{aminobenzoic}$ acid transfer was obtained. The effect of fatty acids was more marked at pH 6.5 than at pH 4.5. In contrast, upon the addition of fatty acids to intestinal lipid membranes which originally contained fatty acids, the permeability coefficient of $\text{p-}\text{aminobenzoic}$ acid tended to decrease, though the permeability through intestinal lipid membranes was larger than that of phosphatidylcholine membranes. The permeability of $\text{p-}\text{aminobenzoic}$ acid across bilayer lipid membranes from intestinal phospholipids was significantly decreased to about equal that of phosphatidylcholine membranes, and reverted to the value of intestinal lipid membranes when fatty acids were added to intestinal phospholipids. It seemed reasonable to assume that free fatty acids in the intestinal neutral lipid fraction could contribute to the increase in the permeability of $\text{p-}\text{aminobenzoic}$ acid. On the basis of above results, possible mechanisms for good absorbability of weakly acidic drugs from the intestine are discussed.     

The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

A new class of lipoxygenase inhibitor. Polyunsaturated fatty acids containing sulfur

A series of unsaturated and polyunsaturated fatty acids with a sulfur atom substituting for a methylene unit of the chain has been prepared and characterized. The syntheses were accomplished by the Wittig coupling of the ylid derived from the triphenylphosphonium salt of 9-bromononanoic acid with aldehydes containing sulfur. The newly formed double bond had predominately the natural Z geometry even when the starting aldehyde was conjugated with the sulfur atom. The sulfides 13-thia-9(Z)-octadecenoic acid ($\text{2}$), 13-thia-9(Z), 11(E)-octadecadienoic acid ($\text{5}$) and 13-thia-9(E), 11(E)-octadecadienoic acid ($\text{6}$) were readily converted into their sulfoxide derivatives by treatment with an equivalent amount of m-chloroperoxybenzoic acid. The structures of the novel compounds were confirmed by the application of ir, uv, ¹H-nmr, ¹³C-nmr, and (as methyl esters) chemical ionization mass spectrometry. Two members of this new family of fatty acids ($\text{5}$ and $\text{6}$) were found to inhibit the catalysis of the oxygenation of linoleic acid by soybean type-1 lipoxygenase. The analysis of the kinetic data for compound $\text{5}$ indicated that the type of inhibition was reversible competitive with an inhibition constant of 30 μM.     

The occurrence of ethanolamine and galactofuranosyl residues attached to Penicilliumcharlesii cell wall saccharides

$\text{Penicillium}$$\text{charlesii}$ incorporates ³H or ¹⁴C from ³H- or ¹⁴C-labeled ethanolamine into an -alkali soluble, alcohol -insoluble fraction obtained from cell walls. Dansyl ethanolamine was isolated from this alcohol-insoluble fraction following dansylation and hydrolysis. The alcohol-insoluble material was non-dialyzable and contained galactofuranosyl, glucosyl, phosphoryl, amino acyl and variable quantities of uronosyl residues. The lack of detectable quantities of mannosyl residues in this material suggests that the galactofuranosyl-containing cell wall polymer is distinct from the peptidophosphogalactomannan which is obtained from culture filtrates of $\text{P}$. $\text{charlesii}$ (Gander $\text{et}$$\text{al}$., (1974) J. Biol. Chem. $\text{249}$, 2063).     

Studies on the mitogenic principle of the lipoprotein from the outer membrane of Escherichia coli

Lipoprotein, from the outer membrane of $\text{E. coli}$, and several derivatives were investigated for lymphocyte stimulating activity in different species. We could show that lipoprotein exhibits activity towards mouse and rat splenocytes and rabbit and bovine lymph node cells; human peripheral blood lymphocytes showed a weak but significant stimulation. Thymocytes of all species were also weakly activated. Altered molecular structures at the C-terminal end of lipoprotein had only small influence on activity, hydrolysis of N-terminal fatty acids abolished mitogenicity.