The preparation,properties and action on Staphylococcus aureus of purified fractions from the cationic proteins of rabbit polymorphonuclear leucocytes.

A micropreparative electrophoresis system for purifying the major staphylocidal fractions of cationic proteins from rabbit polymorphonuclear leucocytes is described. The most staphylocidal fraction prepared is also the most cationic and contains two bands migrating immediately behind protamine sulphate on analytical acid gel electrophoresis. SDS gel electrophoresis indicates that these proteins have low molecular weights between 3,500 and 14,400. The staphylocidal activity of the fraction is affected in the same manner as a crude extract of rabbit PMN granules by iron compounds, respiratory inhibitors, and compounds affecting energy transfer and oxidative phosphorylation. It is stable to heating up to 80 degrees and amino acid analysis shows that it contains 24% arginine. Electron microscopy of staphylococcal spheroplasts treated with the purified fraction or with the crude extract shows that they both have a very marked "blebbing" and distorting action on the double membrane. Comparisons are made between the action of the purified fraction and protamine, and it is concluded that they have very similar, although not identical, properties and actions on staphylococci.     

Microbial adhesion of Cryptosporidium parvum: identification of a colostrum-derived inhibitory lipid

We previously described an unidentified lipid purified from calf small intestine that inhibits the in vitro adhesion of Cryptosporidium parvum sporozoites to host cells [Johnson JK, Schmidt J, Gelberg HB, Kuhlenschmidt MS. Microbial adhesion of Cryptosporidium parvum sporozoites: purification of an inhibitory lipid from bovine mucosa. J Parasitol 2004;90:980-90]. Intestinal mucosa from some calves, however, failed to yield this bioactive lipid. Accordingly, we examined other potential sources, especially dietary sources, of the inhibitory lipid and discovered it was principally derived from bovine colostrum. Interestingly, fresh colostrum yielded little or no inhibitory lipid, however, the lipid was found in relatively large quantities following incubation of colostrum with the aqueous fraction of calf intestinal contents. Using FAB-MS and NMR analysis, the sporozoite inhibitory lipid (SIL) was identified as oleic acid, a monounsaturated fatty acid likely released from colostrum triglycerides and phospholipids by digestion in the lumen of the calf small intestine. Oleic acid dose-dependently inhibited in vitro sporozoite-host cell adhesion with an inhibitory constant (IC(50)) of approximately 5 microM. Comparison of oleic acid with other C-18 fatty acids revealed linolenic, but not stearic acid, also displayed potent inhibitory activity. Neither linolenic nor oleic acid, however, affect either sporozoite or host cell viability at concentrations that inhibit sporozoite adhesion. These results suggest certain colostrum-derived long-chain fatty acids may serve as natural inhibitors of the early steps in C. parvum sporozoite-host cell interactions.     

Ultrastructure, fatty acid content, and metabolic activity of foam cells and other fractions separated from rabbit atherosclerotic lesions

Foam cells and other fractions, separated after incubation of atherosclerotic aortas from cholesterol-fed rabbits with collagenase and elastase, have been investigated with respect to their ultrastructure, cholesterol ester and phospholipid fatty acid composition, DNA content, and metabolic activity as indicated by the uptake and incorporation of ¹⁴C-labeled oleic acid into combined lipid. These studies were concerned with obtaining some information regarding the structural integrity and nature of the isolated foam cells obtained by this method and with determining whether significant breakdown of such cells occurred during the isolation procedure. The foam cell fraction contained typical foam cells with numerous large lipid vacuoles. All cells contained some evidence of myofilaments. The particle fraction (25,000g, 15-minute deposit) consisted disrupted cell organelles, while the remaining fractions appeared to contain more free lipid and liposomes. Both the cholesterol ester and phospholipid fatty acid composition of the particles resembled that of the cells. When ¹⁴C-labeled oleic acid was incubated with the various fractions, significant metabolic activity with respect to uptake of oleic acid and its incorporation into cholesterol ester and phospholipid occurred in the particle fraction, but not in the lipid skin fraction (25,000g, 15-minute floating fat layer). Determination of the DNA content of the cells and other fractions indicated that of the DNA present in the original intima, less than 20% was recovered in the foam cells isolated. The data obtained were consistent with the conclusion that the cells isolated were structurally intact, consisted primarily of myointimal cells and that significant breakdown of foam cells had occurred during the isolation procedure.     

Legionella pneumophila growth restriction and cytokine production by murine macrophages activated by a novel Pseudomonas lipid A.

Peritoneal exudate macrophages from A/J mice activated by purified lipid A preparations from Pseudomonas vesicularis, which contain 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester as the lipid A backbone, restricted the growth of Legionella pneumophila, an intracellular opportunistic bacteria which readily grows in otherwise permissive macrophages from susceptible A/J mice and induced production of the proinflammatory cytokines interleukin 1 and tumor necrosis factor alpha. Activation of the macrophages was similar to that which occurred after stimulation with more conventional lipid A from other bacteria such as salmonellae. A purified fraction A3 preparation from the Pseudomonas lipid A, which lacked only 1 mol of amide-linked fatty acid, in comparison with another fraction (A2), which contained the fatty acid, also markedly activated the usually permissive macrophages from susceptible A/J mice to resist growth of the legionellae. The fraction A3 also induced both interleukin and tumor necrosis factor alpha. These results show that this novel lipid A from P. vesicularis can activate macrophages to resist infection with an opportunistic bacterium in a manner similar to that induced by conventional enterobacterial lipid A and that the hydrophobic portion of this Pseudomonas molecule may have an important role in activation of macrophages.     

Lipid composition and activity of a lytic factor isolated from Plasmodium berghei.

A fraction was obtained from Plasmodium berghei which induced hemolysis of the erythrocytes of mice and hamsters. This fraction, called lytic factor (LF), was found to be composed of a large amount of lipid material. An examination of the lipids showed the major lipids to be monoglycerides, diglycerides, triglycerides, fatty acids, long-chain alcohol, sterol, sterol ester, sterol glycoside, and two cerebrosides. The most abundant component found in the LF was sterol ester, followed in order by cerebrosides, sterol, and sterol glycoside. Lytic activity was found to be lost when samples were boiled for 5 min. An examination of the lipid composition of LF before and after boiling showed changes which may be useful in studies on the mechanism of activity of this factor. The fatty acid composition of the total lipid fraction of LF was examined by gas-liquid chromatography. The major fractions were 18:1 and 16:0 in unheated LF and 16:0 in the heated LF.     

Lipid composition and metabolic activity of subcellular fractions isolated from rabbit atherosclerotic lesions

Homogenates of atherosclerotic rabbit intima were fractionated by ultracentrifugation at 104,000g into three fractions, a lipid skin, a clear supernatant and a particulate fraction. Studies on the distribution and fatty acid composition of the lipid in these fractions demonstrated the existence of several lipid pools, with distinct chemical compositions, in the atherosclerotic intima. The lipid skin was found to be composed predominantly of cholesteryl ester with little free cholesterol or phospholipid. The particulate fraction was low in cholesteryl ester and high in free cholesterol and phospholipid. The cholesteryl ester in the lipid skin was higher in cholesteryl oleate and lower in cholesteryl linoleate than that in the particulate or supernatant fractions. The cholesteryl ester fatty acid pattern of the supernatant was similar to that of the plasma cholesteryl ester. The fatty acid patterns of the phospholipids in all three fractions were similar but significantly different from that of the plasma phospholipids.Following in vitro labeling of the intact intima with (1-¹⁴C) oleic acid prior to fractionation, most of the label incorporated into phospholipid was associated with the particulate fraction. In contrast the label incorporated into cholesteryl ester was found almost exclusively in the lipid skin fraction. Short-term incubation studies failed to demonstrate any movement of labeled cholesteryl ester from one fraction to another. Similar findings both with regard to chemical composition and to incorporation of ¹⁴C-oleic acid into phospholipid and cholesteryl ester were present for the arch, thoracic and abdominal sections of the aorta.     

Characterization of Lipids Associated with Macromolecules of the Intercellular Matrix of Human Aorta

Macroscopically lesion-free parts of human aortas with no or light lesions (group I) and with advanced atherosclerotic lesions (group II) were submitted to a series of successive extractions in order to “solubilize” all the macromolecular components of the arterial wall (“chemical dissection”). Lipids were extracted with methanol-chloroform from all these macromolecular fractions and analyzed for cholesterol, triglycerides, phospholipids. The fatty acid composition of the separated fractions was determined by GLC. The lipid composition and fatty acid spectrum of the macromolecular fractions of group I and group II aortas was compared.

Total lipids increased in the freely extractable (“non associated lipids, ～ 78 % of total) fraction as well as in the fraction “associated” with collagen and elastin. Free and esterified cholesterol increased also both in the “freely extractable” and in the collagen-elastin-associated lipids”, ～ 78 % of total) fraction as well was higher (+ 100 %) than that of free cholesterol (+ 60%).

Triglycerides increase also by 15 to 70 % in all fractions except in the elastin-associated fraction.

Free fatty acids increased by 40 to 400 % in all extracts associated with macromolecular fractions but not in the “freely extractable” fraction where they decreased. Phospholipids show less marked variations (～ 10 %) and decrease in the elastin associated lipids of group II aortas.

The fatty acid spectrum of group II lipids associated with macromolecules differs from that of group I. There is a relative increase of longer chains (C > 18, especially 20: 1 and 20: 2 acids). No such increase in the “long” fatty acids was seen in the “freely extractable” lipid fraction.

Elastin isolated from group II aortas is significantly enriched in total lipids, cholesterol (free and esterified) and free fatty acids and contains the widest spectrum of fatty acids (from 11:2 to 22: 1) with a significant fraction of total fatty acids as “odd” carbon chains.

There appears to be a correlation between the decrease of triglyceride-bound fatty acids and the increase of free fatty acids. The free fatty acid concentration exceeds both in group I and II aortas the concentration of fatty acid esters. This increase in free fatty acids “associated” with intercellular matrix macromolecules and especially with elastin may be the result of an increased hydrolysis of esters and/or a decreased esteri-fication in advanced atherosclerotic aortas. The accumulation of long chain and “odd” fatty acids in elastin may be an important factor in its accelerated degradation during the atherosclerotic process.     

Characterization of lipids associated with macromolecules of the intercellular matrix of human aorta.

Macroscopically lesion-free parts of human aortas with no or light lesions (group I) and advanced atherosclerotic lesions (group II) were submitted to a series of successive extractions in order to "solubilize" all the macromolecular components of the arterial wall ("chemical dissection"). Lipids were extracted with methanol-chloroform from all these macromolecular fractions and analyzed for cholesterol, triglycerides, phospholipids. The fatty acid composition of the separated fractions was determined by GLC. The lipid composition and fatty acid spectrum of the macromolecular fractions of group I and group II aortas was compared. Total lipids increased in the freely extractable ("non associated lipids, approximately 78% of total) fraction as well as in the fraction "associated" with collagen and elastin. Free and esterified cholesterol increased also both in the "freely extractable" and in the collagen-elastin-associated lipids", approximately 78% of total) fraction as well was higher (+ 100%) than that of free cholesterol (+ 60%). Triglycerides increase also by 15 to 70% in all fractions except in the elastin-associated fraction. Free fatty acids increased by 40 to 400% in all extracts associated with macromolecular fractions but not in the "freely extractable" fraction where they decreased. Phospholipids show less marked variations (approximately less than 10%) and decrease in the elastin associated lipids of group II aortas. The fatty acid spectrum of group II lipids associated with macromolecules differs from that of group I. There is a relative increase of longer chains (C greater than 18, especially 20:1 and 20:2 acids). No such increase in the "long" fatty acids was seen in the "freely extractable" lipid fraction. Elastin isolated from group II aortas is significantly enriched in total lipids, cholesterol (free and esterified) and free fatty acids and contains the widest spectrum of fatty acids (from 11:2 to 22:1) with a significant fraction of total fatty acids as "odd" carbon chains. There appears to be a correlation between the decrease of triglyceride-bound fatty acids and the increase of free fatty acids. The free fatty acid concentration exceeds both in group I and II aortas the concentration of fatty acid esters. This increase in free fatty acids "associated" with intercellular matrix macromolecules and especially with elastin may be the result of an increased hydrolysis of esters and/or a decreased esterification in advanced atherosclerotic aortas. The accumulation of long chain and "odd" fatty acids in elastin may be an important factor in its accelerated degradation during the atherosclerotic process.     

Staphylocidal action of thrombin-induced platelet microbicidal protein is influenced by microenvironment and target cell growth phase.

Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic peptide released from rabbit platelets following exposure to thrombin in vitro. This peptide exerts potent in vitro microbicidal activity against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. It is known that the microbicidal actions of other cationic antimicrobial peptides (e.g., neutrophil defensins) are influenced by environmental factors and target cell growth phase. However, whether these parameters affect tPMP microbicidal activity has not been studied. Thus, we assessed the in vitro bactericidal activity of tPMP against two tPMP-susceptible strains, Bacillus subtilis ATCC 6633 and S. aureus 502A, in various target cell growth phases or under various microenvironmental conditions. The conditions studied included differing bacterial growth phase (logarithmic versus stationary), temperature (range, 4 to 42 degrees C), pH (range, 4.5 to 8.5), cationicity (range, 0.1 mM to 2 M), anionicity (range, 0.08 to 5 microM), and neutral carbohydrates ranging in molecular weight (MW) from 180 to 37,700 (range, 50 to 500 mM) as well as rabbit platelet-free plasma and serum. tPMP staphylocidal activity was greater against logarithmic- than stationary-phase cells. tPMP bactericidal activity against both B. subtilis and S. aureus was directly correlated with temperature and pH, with microbicidal activity exhibited near the physiological range (37 to 42 degrees C and pH 7.2 to 8.5, respectively). The presence of cations (Na+, K+, Ca2+, and Mg2+) decreased tPMP bactericidal activity in a time- and concentration-dependent manner, with complete inhibition at monovalent or divalent cation concentrations of > or = 250 or > or = 10 mM, respectively. Staphylocidal activity of tPMP was also inhibited by the polyanions polyanetholsulfonic acid and polyaspartic acid, at 0.1 and 0.4 microM, respectively. Coincident exposure with low-MW carbohydrates (glucose, sucrose, and melezitose) did not affect tPMP staphylocidal activity. However, higher-MW carbohydrates (raffinose and dextrans) decreased tPMP activity in a manner directly proportional to their concentration and MW. Solute-mediated inhibition of tPMP bactericidal activity was independent of solute osmolality but directly related to the duration of tPMP-solute coexposure. tPMP enhanced the staphylocidal activities of platelet-free plasma and heat-inactivated serum, while the activity of normal serum was not affected. These collective observations suggest that tPMP retains antimicrobial activities under physiological conditions which are likely to be relevant to host defense in vivo.     

Lipid-free glycerol teichoic acids with potent membrane-binding activity.

Lipid analysis of several glycerol teichoic acid preparations strongly indicated that covalently bound lipid is not required for spontaneous adsorption of glycerol teichoic acid to erythrocyte membranes. Although fatty acids were detected in each of four batches, none were covalently bound. Chloroform-ether-extracted antigens retained potent erythrocyte membrane-binding activity as measured by passive hemagglutination, even though they were shown to contain less than one fatty acid residue per 4,869 teichoic acid chains. Mild ammonolysis abolished erythrocyte-sensitizing activity in passive hemagglutination, but further studies indicated the loss of activity was due to partial destruction of the polyglycerophosphate backbone and not to the removal of esterified lipid. The amount of hydrolyzed antigen required to produce 100% passive hemagglutination inhibition was between 170 and 330 times the amount required to produce the same result using unhydrolyzed glycerol teichoic acid. The average chain length was reduced from 19.1 to 9.7, 7.4, and 5.1 glycerophosphate residues for antigen samples hydrolyzed for 1, 5, and 16 h, respectively.     

Phytochemical and biological investigations of Atriplex semibacata R .BR. growing in Egypt

The lipid content of Atriplex semibacata growing in Egypt was studied. The unsaponifiable fraction was identified by GLC. A series of hydrocarbons ranging from C(14)- C(28) in addition to cholesterol, stigmasterol and the triterpenoids α and β - amyrin were identified. GLC analysis of fatty alcohols fraction revealed the presence of six fatty alcohols in which dotriacontanol (C(32)H(66)O) was the major (14.68%). Six compounds (five coumarins and one phenolic acid) were isolated for the first time from A. semibacata. The coumarin constituents isolated from the chloroform and the ethyl acetate fractions of the aqueous alcoholic extract of A. semibacata were identified as scopoletin, umbelliferorne, coumarin, scopolin, 7-methoxy coumarin in addition to a phenolic acid P-coumaric acid. Also, the flavonoidal compounds isolated from the n-butanol fraction of the plant revealed the presence of kaempferol 3-O glucoside and acacetin. Their identity was proved by m.p., TLC, PC, UV and MS analysis. The alcohol extract showed significant antimicrobial activity against G-ve bacteria, moderate activity against G+ve bacteria. On the other hand, the pet. Ether extract showed marked activity against G+ve bacteria and fungi, also the G-ve bacteria was greatly inhibited by the chloroform extract. The different extracts of the plant exhibited no cytotoxic activity against Erlich-ascites carcinoma cells line at the tested concentrations, also showed a strong antioxidant activity using DPPH.     

Lipid components of the hydrocarbon assimilating yeast Candida lipolytica (strain 10)

The utilization of n-hexadecane by Candida lipolytica (strain 10) was studied with respect to the lipid content, phospholipid and fatty acid profiles resulting at various growth times. Thin layer chromatography of the lipid extracts showed quantitative changes in the different lipid classes. The phospholipid fraction obtained at each growth time was separated into 8 classes: lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, glycophospholipid, phosphatidylglycerol, cardiolipin, and phosphatidic acid. Differences in the percentage fatty acid composition of the lipid extracts were observed at various stages of growth. The cellular fatty acids included palmitic, palmitoleic (35–52%), stearic, oleic, linoleic (26–39%), and pentadecanoic (2–12%) as major components. This indicates that fatty acid(s) of the same length as that of the substrate was the most abundant component, thus showing intact incorporation mechanism. Fatty acids having longer chain lengths were also formed in substantial amounts indicating C₂ addition and β-oxidation of the fatty acids formed in the yeast.     

Lysosomal hydrolysis of lipids in a cell culture model of smooth muscle foam cells

Rabbit aortic smooth muscle cells take up lipid droplets when they are presented using an inverted culture technique. These droplets were localized in secondary lysosomes as demonstrated by staining for acid phosphatase. Initially, 69% of the cell volume was occupied by lipid, and 94% of the lipid was in lysosomes. After a 24-hr clearance period, the cell volume occupied by lipid decreased to 53%, although there was no change in the fraction of cell lipid that was in lysosomes. To confirm that hydrolysis of droplet lipid was occurring in lysosomes, cultures were exposed to medium containing Sandoz 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, for 24 hr in the presence and absence of chloroquine, ammonium chloride, or methylamine. Although the hydrolysis of cholesteryl oleate was sensitive to these lysosomotropic agents, the hydrolysis of triolein was not. Using reconstituted LDL containing cholesteryl oleate and triolein, we demonstrated that the hydrolyses of cholesteryl oleate and triolein were equally sensitive to the lysosomotropic agents when the cells were not loaded with droplet lipid. However, in cells loaded with lipid, hydrolysis of LDL cholesteryl ester was sensitive to the lysosomotropic agents but hydrolysis of triolein was not. We therefore conclude that both droplet lipids were hydrolyzed in lysosomes, and we attribute the failure of the lysosomotropic agents to inhibit fully the hydrolysis of droplet triolein to the presence of a large mass of free fatty acids in the lysosome that maintains a sufficiently low pH to sustain the triglyceridase activity, but not the cholesteryl esterase activity, of the lysosomal acid lipase.     

Observations on the lipids ofOochoristica agamae (Cestoda)

An investigation of the lipids ofOochoristica agamae, an anoplocephalid cestode of theAgama lizard, was undertaken. Total lipids of the parasite accounted for 8.4% of the fresh weight; neutral lipids comprised 82.98% of the total, glycolipids, 5.01%, and phospholipids, 12.03%. The major lipid classes inO. agamae include triglycerides, cholesterol, phosphatidyl choline, and phosphatidyl ethanolamine. The 16-and 18-carbon fatty acids were predominant in the parasite. Hexadecenoic acid, usually found at low concentrations in the lipids of helminth parasites, was the most abundant of the 16-carbon fatty acids ofO. agamae (notably in the neutral lipid fraction). Although octadecatrienoic acid occurred only in trace amounts in the intestinal contents of the host, significant amounts of this fatty acid were detected in the parasite. A lack of 20-carbon fatty acids was determined in the lipids of the host's intestinal contents and the neutral lipid fraction of the parasite.O. agamae is suspected to be capable of modifying fatty acids obtained from dietary sources by chain elongation.     

Characterization of a novel thioesterase (PtTE) from Phaeodactylum tricornutum

The Phaeodactylum tricornutum (P. tricornutum) thioesterase PtTE was encoded by a 648 bp open reading frame. The deduced 216 amino acids showed no similarity with plant acyl-acyl carrier protein (ACP) thioesterases and bacterial thioesterases. Southern blot analysis revealed that one copy of PtTE was present in the P. tricornutum genome, and Real-time quantitative PCR showed that PtTE was up-regulated upon nitrogen deprivation. Thioesterase activity of PtTE was established by heterologous expression of PtTE cDNA in Escherichia coli (E. coli) XL1-Blue and K27fadD88, a mutant strain of fatty acid β-oxidation pathway. The substrate specificity of PtTE was determined by fatty acid profile analyses of the culture supernatant and membrane lipid of recombinant strains. Recombinant PtTE in E.coli enhanced total fatty acid content of XL1-Blue by 21%, and also changed the fatty acid compositions of membrane lipid and culture supernatant. These changes were directed predominantly towards C18:0 and C18:1 fatty acids. Overexpression of PtTE alone in P. tricornutum did not alter the fatty acid composition of P. tricornutum, but enhanced total fatty acid content by 72%. This novel thioesterase gene shows its potential in metabolic engineering for enhancing lipid yield of microalgae. This is so far the first report of thioesterase from eukaryotic microalgae.     

Free fatty acids released from phospholipids are the major heat-stable hemolytic factor of Entamoeba histolytica trophozoites.

The major hemolytic activity of Entamoeba histolytica trophozoites is located in a vesicular fraction called P30 and known to be due to heat-labile and heat-stable hemolytic components whose effect increases up to 100 times during preincubation at 36 degrees C. The heat-stable hemolytic activity (HSHA) was found in the chloroform-methanol extract of preincubated P30, whose partition with 2 M KCl yielded a lipid fraction, an interphase, and an aqueous phase. HSHA was detected only in the lipid fraction, where it amounted to 59% of the chloroform-methanol extract activity and increased 50% when supplemented with the interphase material; it was accounted for by the free fatty acids, whose potency increased 33% with the interphase material, and was blocked by delipidated bovine serum albumin. A parallel increase in free fatty acids and lysophospholipids and a corresponding decrease in phospholipids were observed during P30 preincubation. Most of the phospholipase activity of trophozoite homogenates was also found in P30. Therefore, most of the HSHA generated during preincubation was due to free fatty acids released from phospholipids by a P30 phospholipase that may contribute significantly to E. histolytica cytopathogenicity and virulence.     

Changes in lipid composition of neuroblastoma C1300 N18 cell during differentiation

Phospholipids and cholesterol were found to be the main lipids in mature and immature neuroblastoma cells. The ratios for the total cholesterol/phospholipids in these undifferentiated and differentiated cells were 0.33 and 0.52, respectively. The ratios of 0.45 and 0.62 were obtained with corresponding plasma membrane fractions. Individual fatty acid contents in the loosely bound lipid fraction were higher than in tightly bound lipids. The total levels of saturated fatty acids increased in both of these fractions. While arachidonic acid content significantly decreased, it increased simultaneously (600%) in the free fatty acid fraction during differentiation. The amount of cholesterol esters increased three-fold as a result of maturation. For the first time it was possible to detect, in neuroblastoma cells, several lipids, namely N-acylphosphatidylethanolamine, N-acylethanolamine and semilysobisphosphatidic acid. They all changed during maturation. Total N-acylphosphatidylethanolamine content decreased by 50%, disappearing completely from membrane fractions. N-Acylethanolamine disappeared from the cell as well as from membrane fractions. On the other hand the total cellular content of semilysobisphosphatidic acid increased without any alterations in its membrane content. Functional implications of our investigations are discussed.     

Chronic alcoholic skeletal muscle myopathy: a clinical, histological and biochemical assessment of muscle lipid.

Muscle biopsy samples were analysed from five control subjects, four patients with mild to moderate fibre atrophy and four patients with severe atrophy. Patchy increase in lipid was noted with oil red O staining but there was no consistent association of lipid with selective fibre types. Ultrastructural studies demonstrated lipid droplets both subjacent to the sarcolemma and between fibrils. Quantitative analysis showed that the increased lipid was solely due to excess triglyceride. GLC analysis of free and esterified acids was performed. The profiles were essentially similar for the phospholipid and free fatty acid fractions. The triglyceride fraction showed a decrease of myristate, stearate and linoleate with an increase in oleate and arachidate in the alcoholic tissue compared with control. The cholesteryl ester fraction showed an increase in palmitate with a decrease in stearate and oleate in the alcoholic muscle. The accumulation of lipid correlated with mean daily alcohol consumption but not with degree of atrophy suggesting that the two processes probably had different pathogenic mechanisms.     

Staphylocidal action of thrombin-induced platelet microbicidal protein is not solely dependent on transmembrane potential.

Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic, antimicrobial peptide released from rabbit platelets when stimulated with thrombin. We studied the relationship between staphylococcal transmembrane potential (delta psi) and tPMP staphylocidal activity. A genetically related pair of Staphylococcus aureus strains, 6850 and JB1, which differ in delta psi generation (-143 and -97 mV, respectively) were used. Mutant JB-1 was substantially less susceptible to tPMP than the parental strain, 6850. Menadione supplementation, which normalized the delta psi of strain JB-1, did not restore JB-1 tPMP susceptibility. These findings suggest that the staphylocidal activities of tPMP require factors other than or in addition to an intact delta psi.     

Assessment of oral ascorbate in three children with chronic granulomatous disease and defective neutrophil motility over a 2-year period

Two brothers and their sister with chronic granulomatous disease, elevated levels of serum IgE and defective neutrophil motility were treated with a single oral daily dose of 1 g sodium ascorbate as a supplement to prophylactic trimethoprim--sulphamethoxazole therapy for 2 years. Laboratory tests of neutrophil functions were performed prior to ascorbate therapy and repeated at 1-monthly intervals for 6 months and at 6-monthly intervals thereafter. Introduction of ascorbate to the therapeutic regimen was accompanied by slight increases in neutrophil hexose monophosphate shunt activity and staphylocidal activity and good improvement of neutrophil motility in all three children. The improved staphylocidal activity was not due to ascorbate-mediated inhibition of neutrophil or serum catalase activities or to detectable increases in superoxide and H2O2 production or activity of the MPO/H2O2/halide system. Both male children have remained free from obvious infection since ascorbate was added to their therapeutic regimen; their sister has experienced one urinary tract infection during a period when treatment with prophylactic co-trimoxazole and ascorbate was inadvertently stopped. All three children have gained weight.