Enhancement of the autocatalytic activation of trypsinogen to trypsin by bile and bile acids

The activation of trypsinogen to trypsin in the small intestine can occur by the action of enterokinase or, alternatively, as an autocatalytic process catalysed by trypsin itself. We have found that bile salts and human bile cause a significant enhancement of the autocatalytic activation of trypsinogen. This effect is dependent on the calcium ion concentration and is most marked around pH 5.4 and 7.8. An optimum concentration exists for each bile salt at which the greatest enhancement occurs. At this concentration, certain bile salts have been shown to produce activation effects of up to 55-fold. It is suggested that this activation of the autocatalytic process by bile plays an important role in protein digestion in the small intestine, since it has been shown previously that duodenal trypsin levels are abnormally low in patients with an impairment of bile secretion.     

Sex-linked differences in bile acid metabolism of germfree rats

The bile acid composition was investigated in male and female germfree rats. β-Muricholic acid and cholic acid were the major bile acids in both sexes; in addition, 3β-hydroxy-5-cholenoic acid, chenodeoxycholic acid, α-muricholic acid, allochenodeoxycholic acid and allocholic acid were present. Important sex-linked differences in the relative amounts and the sulfation of these substances were observed. β-Muricholic and cholic acid accounted for 61.4 % and 27.7 % of total bile acids in the small intestine of males; females had 38.9 % of β-muricholic acid and 50 % of cholic acid. In females, the bile acid sulfate fraction increased from 1.1 % in the small intestine to 22.3 % in the large intestine; in males these values were 0.2 % and 1.7 %, respectively. A considerable increase in the relative amounts of allochenodeoxycholic and allocholic acid was observed in the cecum and large intestine of the female rat, where more than 70 % of these substances was in the bile acid sulfate fraction. In males these allo-bile acids were mainly in the unsulfated fraction and their relative amounts did not increase in the large intestine.     

Bile secretion in the chicken: effects of secretin, cholecystokinin- pancreozymin and duodenal mucosal extract

The effects of i.v. administration of secretin, CCK-PZ, acid extracts from the duodenal mucosa and the duodenal acidification of the intestine on bile secretion were studied in anaesthetized chickens. Secretin and acid extracts from the duodenal mucosa, which increase bile flow, caused comparable modifications in bile composition; infusion of HCl to the duodenum only induced slight modifications. CCK-PZ caused a pronounced cholecystokinetic effect and, to a lesser degree, it also showed choleretic effects. The results suggest that in the hormonal regulation of bile secretion in the chicken CCK-PZ is more important than secretin and furthermore that the choleretic activity of the latter must be carried out by other secretin-like peptides.     

古细菌极性脂质片段的提取和四醚脂质体的制备及稳定性研究

脂质体作为药物口服载体,面临的主要障碍之一是其在肠道胆汁盐和肠道酶的作用下极不稳定,造成包裹药物的损失和破坏。研究中,应用丙酮沉淀、薄层制备层析以及甲醇沉淀相结合的方法,从培养的古细菌Sulfolobus acidocaldarius中分离和纯化极性脂质抽提片段(polarlipid fraction extract,PLFE),制备了由PLFE为组分的四醚脂质体;并着重评估其在模拟肠液中的稳定性。结果显示:改进的提取方法操作简单、有效;在模拟人肠道环境的体外稳定性实验中,四醚脂质体的稳定性明显优于普通磷脂脂质体。这些结论表明,具有独特结构的极性脂质组成的四醚脂质体,具备作为药物的口服输送载体的潜力。     

The effect on gastrointestinal motility after ingestion of raw bile of grass carp (Ctenopharyngodon idellus) in the conscious rats

The influence of raw grass carp bile on gastrointestinal motility was studied in conscious rats. It was found that the toxic bile prolonged the gastric emptying time, increased a marker substance traversed along the small intestine and accelerated the transit time along the colon. These findings might be explained by a very efficient protective response of the digestive system, and/or poisoning effect of the invading toxic substance in rats. Accumulation of fluid was found in the stomach, 20 min after ingestion of 0.3 ml of the toxic bile. Besides, ingestion of the raw bile also increased the urea nitrogen in blood, which may result from a loss of fluid volume through the gastrointestinal tract and (or) a direct action of the bile on the kidneys.     

Bile acids in the rat: studies in experimental occlusion of the bile duct

Bile acids in the plasma, urine, and small intestine of adult male rats with occluded bile ducts have been studied using a method of high specificity for their determination. After bile duct ligation cholic acid rapidly accumulates in the plasma for 8 hr, remains high for a further 8 hr, and subsequently diminishes; bile acids disappear from the small intestine. During the first 12 hr after bile duct ligation the excretion of trihydroxy acids in the urine was 10 times that of the dihydroxy acids. Subsequently the two excretion rates became equal. Because bile acids have been implicated in the etiology of hepatic damage following bile duct ligation, studies have been made of the effect on the liver of removing (with cholestyramine) and supplementing (with cholic acid) the intestinal bile acid pool. The addition of cholestyramine to the stock diet prevented the rise in trihydroxy bile acids after bile duct ligation, but did not prevent the development of histological abnormalities in the liver. Supplementing the diet with cholic acid raised the plasma cholic acid levels but had little effect on the hepatic histological findings.     

Characterization of liposomes coated with S-layer proteins from lactobacilli

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.     

Further studies on the properties of bile acids. II. Effect of bile acids on the reactivity of adrenergic and cholinergic receptors in rat intestine

The effects of bile acids (deoxycholic, cholic and dehydrocholic) were studied on the action of certain autonomic system drugs (isoprenaline, adrenaline, noradrenaline and acetylcholine). It was also tried to explain the causes of these changes in the light of the results of experiments with the widely used antagonists of adrenergic and cholinergic receptors. The experiments were carried out on isolated rat intestine by the method of Magnus. It was found that bile acids decreased the relaxing effect of isoprenaline and caused inversion of the action of adrenaline and noradrenaline on the intestine. Changes in the action of catecholamines are caused by stimulation of alpha-adrenergic receptors enhanced by bile acids, with a simultaneous decreased stimulation of beta-receptors. Bile acids cause also an increase of the effect of acetylcholine on the intestine.     

Determination of liposome size: a tool for protein reconstitution

Reconstitution of proteins into liposomes is a widespread approach to analyzing their biological function. Many protocols exist for this procedure and for the subsequent analysis of proteins. Here, we establish a procedure for preparation and analysis of liposomes with a lipid composition reflecting the outer envelope of chloroplasts. First, the stability of the liposomes in different buffer systems was investigated to provide information for the storage of the reconstituted system. Then, the size of the liposomes created by filtration through a polycarbonate filter dependent on the lipid composition was analyzed. Subsequently, solubilization of the liposomes composed of lipids with the outer envelope composition by dodecylmaltoside and octylglucoside as a preceding step of reconstitution was studied. Finally, we developed a straightforward method to determine the size of liposomes by absorption spectroscopy. The described setup allows the construction of reconstitution protocols, including the final determination of the liposome size.     

Characterization of liposomes coated with S-layer proteins from lactobacilli

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.     

Transport of 125I-poly I : poly C incorporated in liposomes from the enteric cavity to the small intestine mucosa. An electron microscopic autoradiographic study

Transport of 125I-poly(I) : poly(C) incorporated into liposomes trough the small intestine mucose was investigated by electron microscopic autoradiography. With the migration of liposomes into the mucous layer on the luminal surface of the intestine up to the glycocalix level of microvilli these undergo degradation with the formation of monolayer liposomes from which polynucleotide is released. Later on the poly(I) : poly(C) or its fragments transported through the enterocytes to be accumulated in cells of the connective tissue stroma of the small intestine mucose. Part of polynucleotide was incorporated up to the arterial and lymphoid capillary level. Apparently, on the way of its transport the polynucleotide is affected by pancreatic and tissue nucleases. The accumulation of polynucleotide in macrophages, fibroblasts, lymphocytes, plasma cells and smooth muscle cells was traced. It is supposed that the polynucleotide accumulated in stroma of the small intestine mucose may preserve its interferon inducing activity.     

Inhibitory effect of cholesterol on the uptake of liposomes by liver and spleen

The effect of cholesterol content of small unilamellar (SUV) and reverse phase (REV) liposomes on blood clearance and tissue distribution has been studied. [¹⁴C]Inulin has been used as an aqueous marker of liposomes to represent the uptake of intact liposomes in tissues. The blood clearance of the intravenously-injected SUV and REV liposomes depends on the cholesterol content of liposomes. The cholesterol-free (0 mol%) liposomes are cleared more readily from the circulation than the cholesterol-poor liposomes (20 mol%) and the cholesterol-poor are cleared more rapidly than the cholesterol-rich (46.6 mol%) liposomes. This clearance pattern of liposomes from the circulation is not attributed to the change of size of liposomes due to the increase in cholesterol content of liposomes. However, poor stability of cholesterol-free or cholesterol-poor liposomes in the circulation is partly responsible, but the predominant factor responsible for the observed blood clearance pattern is the inhibitory effect of cholesterol on the uptake of liposomes by reticuloendothelial-rich tissues liver and spleen. Uptake of liposomes by these organs is decreased with increasing cholesterol content of vesicles. It is suggested that to produce liposome preparations with a long circulating half life in vivo it is necessary to inhibit their uptake by liver and spleen.     

Uptake of small liposomes by non-reticuloendothelial tissues

The distribution of liposomes within the intravascular space and the extent to which they escape into extravascular space strongly impact on the application of lipid vesicles as a carrier for pharmacologically active agents. The present study investigates how intact small unilamellar vesicles (SUV) may be taken up by different tissues after intravenous injection into mice, using various types of SUV with different entrapped markers, lipid composition, size, doses of liposomal lipids and stability in the blood. Our focus was specifically on sphingomyelin (or distearoyl phosphatidylcholine)/cholesterol (2:1, mol/mol) SUV, which are known to be stable in the blood circulation. Our results indicated that, in addition to the reticuloendothelial tissues, intact SUV were taken up in several other parts of the body, including intestine, skin, carcass and legs. It appears that the accumulation of SUV in the intestine and the skin increases with time post-injection. Furthermore, from the kinetic data, the process of uptake of SUV by the skin and intestine is compatible with a non-saturable pathway, which follows first-order kinetics. This suggests that the cells involved in the uptake of SUV in the intestine and skin are not phagocytic cells, which are normally saturable.     

Heterogeneity of early intermediates in cell-liposome fusion mediated by influenza hemagglutinin

To explore early intermediates in membrane fusion mediated by influenza virus hemagglutinin (HA) and their dependence on the composition of the target membrane, we studied lipid mixing between HA-expressing cells and liposomes containing phosphatidylcholine (PC) with different hydrocarbon chains. For all tested compositions, our results indicate the existence of at least two types of intermediates, which differ in their lifetimes. The composition of the target membrane affects the stability of fusion intermediates at a stage before lipid mixing. For less fusogenic distearoyl PC-containing liposomes at 4 degrees C, some of the intermediates inactivate, and no intermediates advance to lipid mixing. Fusion intermediates that formed for the more fusogenic dioleoyl PC-containing liposomes did not inactivate and even yielded partial lipid mixing at 4 degrees C. Thus, a more fusogenic target membrane effectively blocks nonproductive release of the conformational energy of HA. Even for the same liposome composition, HA forms two types of fusion intermediates, dissimilar in their stability and propensity to fuse. This diversity of fusion intermediates emphasizes the importance of local membrane composition and local protein concentration in fusion of heterogeneous biological membranes.     

Archaeobacterial ether lipid liposomes (archaeosomes) as novel vaccine and drug delivery systems

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents. The objective of this article is to review the properties and potential applications of novel liposomes made from the membrane lipids of Archaeobacteria (Archaea). These lipids are unique and distinct from those encountered in Eukarya and Bacteria. Polar glycerolipids make up the bulk of the membrane lipids, with the remaining neutral lipids being primarily squalenes and other hydrocarbons. The polar lipids consist of regularly branched, and usually fully saturated, phytanyl chains of 20, 25, or 40 carbon length, with the 20 and 40 being most common. The phytanyl chains are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). It has been shown only recently that total polar lipids of archaeobacteria, and purified lipid fractions therefrom, can form liposomes. We refer to liposomes made with any lipid composition that includes ether lipids characteristic of Archaeobacteria as archaeosomes to distinguish them from vesicles made from the conventional lipids obtained from eukaryotic or eubacterial sources or their synthetic analogs. In general, archaeosomes demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts, and serum proteins. Some archaeosome formulations can be sterilized by autoclaving, without problems such as fusion or aggregation of the vesicles. The uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of conventional liposome formulations. Studies in mice have indicated that systemic administration of several test antigens entrapped within certain archaeosome compositions give humoral immune responses that are comparable to those obtained with the potent but toxic Freund's adjuvant. Archaeosome compositions can be selected to give a prolonged, sustained immune response, and the generation of a memory response. Tissue distribution studies of archaeosomes administered via various systemic and peroral routes indicate potential for targeting to specific organs. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any noticeable toxicity in mice. The stability, tissue distribution profiles, and adjuvant activity of archaeosome formulations indicate that they may offer a superior alternative to the use of conventional liposomes, at least for some biotechnology applications.     

Monensin intercalation in liposomes: effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells.

Monensin, a carboxylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin (1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charges vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of 125I-gelonin from neutral vesicles is significantly higher (approximately 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.     

Functional and structural changes in the small intestine during experimental hypercholesteremia

Functional and structural changes that occurred in the small intestine and liver of rabbits with alimentary hypercholesterolemia have been studied. Maximal rise of peripheral blood cholesterol after a single exposure to cholesterol was coupled with an increased penetration of chylomicrons and low and very low density lipoproteins from the intestine to the circulation. The decrease of cholesterol excretion along the whole length of the small intestine was accompanied by activation of the release of high density lipoproteins into the blood outflowing from the intestine. The decay of chylomicrons in the liver was restricted during the first days of hypercholesterolemia. Cholesterol concentration in the bile was decreased. The microvessels of the small intestine demonstrated the dilatation of the postcapillary-venular component erythrocyte aggregates and capillarostases.     

Effect of bile anionic polypeptidic fraction on the fate of cholesterol carried by liposomes in the rat

[14C]Cholesterol associated with liposomes with or without anionic polypeptidic fraction was administered intravenously to the rat. The cholesterol originated from liposomes including anionic polypeptidic fraction is secreted in bile much later, is stored in liver in higher quantity, and is metabolized into bile salts in lesser quantity during the 4 hr of experimentation than the cholesterol issued from liposomes exempt of anionic polypeptidic fraction. From these results it can be postulated that the cholesterol associated with liposomes containing anionic polypeptidic fraction might be directed in a particular liver pathway.