Synthesis and characterization of a novel rhodamine labeled cholesterol reporter.

We introduce the novel fluorescent cholesterol probe RChol in which a sulforhodamine group is linked to the sixth carbon atom of the steroid backbone of cholesterol. The same position has recently been selected to generate the fluorescent reporter 6-dansyl-cholestanol (DChol) and the photoreactive 6-azi-cholestanol. In comparison with DChol, RChol is brighter, much more photostable, and requires less energy for excitation, i.e. favorable conditions for microscopical imaging. RChol easily incorporates into methyl-ß-cyclodextrin forming a water-soluble inclusion complex that acts as an efficient sterol donor for cells and membranes. Like cholesterol, RChol possesses a free 3'OH group, a prerequisite to undergo intracellular esterification. RChol was also able to support the growth of cholesterol auxotrophic cells and can therefore substitute for cholesterol as a major component of the plasma membrane. According to subcellular fractionation, slight amounts of RChol (~12%) were determined in low-density Triton-insoluble fractions whereas the majority of RChol was localized in non-rafts fractions. In phase-separated giant unilamellar vesicles, RChol preferentially partitions in liquid-disordered membrane domains. Intracellular RChol was transferred to extracellular sterol acceptors such as high density lipoproteins in a dose-dependent manner. Unlike DChol, RChol was not delivered to the cholesterol storage pathway. Instead, it translocated to endosomes/lysosomes with some transient contacts to peroxisomes. Thus, RChol is considered as a useful probe to study the endosomal/lysosomal pathway of cholesterol.

Side-chain oxysterols modulate cholesterol accessibility through membrane remodeling.

Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are key regulators of cholesterol homeostasis. New evidence suggests that the alteration of membrane structure by 25-HC contributes to its regulatory effects. We have examined the role of oxysterol membrane effects on cholesterol accessibility within the membrane using perfringolysin O (PFO), a cholesterol-dependent cytolysin that selectively binds accessible cholesterol, as a sensor of membrane cholesterol accessibility. We show that 25-HC increases cholesterol accessibility in a manner dependent on the membrane lipid composition. Structural analysis of molecular dynamics simulations reveals that increased cholesterol accessibility is associated with membrane thinning, and that the effects of 25-HC on cholesterol accessibility are driven by these changes in membrane thickness. Further, we find that the 25-HC antagonist LY295427 (agisterol) abrogates the membrane effects of 25-HC in a nonenantioselective manner, suggesting that agisterol antagonizes the cholesterol-homeostatic effects of 25-HC indirectly through its membrane interactions. These studies demonstrate that oxysterols regulate cholesterol accessibility, and thus the availability of cholesterol to be sensed and transported throughout the cell, by modulating the membrane environment. This work provides new insights into how alterations in membrane structure can be used to relay cholesterol regulatory signals.

Structure-activity relationships in aminosterol antibiotics: the effect of stereochemistry at the 7-OH group.

Squalamine and three aminosterol analogs have been shown to inhibit bacterial cell growth and induce lysis of large unilamellar phospholipid vesicles. The analogs differ in the identity of the polyamine attached at C3 of the sterol, and the stereochemistry of a hydroxyl substituent at C7. Analogs with a tetraammonium spermine polyamine are somewhat more active than analogs with a shorter trisammonium spermidine polyamine, and analogs with an axial (α) hydroxyl substituent at C7 are more active than analogs with the corresponding equatorial (ß) hydroxyl group. There is some variability noted; the 7ß-OH spermine analog is the most active compound against Escherichia coli, but the least effective against Pseudomonas aeruginosa. Lytic activity correlates well with antimicrobial activity of the compounds, but the lytic activity varies with the phospholipid composition of the vesicles.

Increased leaf size: different means to an end.

The final size of plant organs, such as leaves, is tightly controlled by environmental and genetic factors that must spatially and temporally coordinate cell expansion and cell cycle activity. However, this regulation of organ growth is still poorly understood. The aim of this study is to gain more insight into the genetic control of leaf size in Arabidopsis (Arabidopsis thaliana) by performing a comparative analysis of transgenic lines that produce enlarged leaves under standardized environmental conditions. To this end, we selected five genes belonging to different functional classes that all positively affect leaf size when overexpressed: AVP1, GRF5, JAW, BRI1, and GA20OX1. We show that the increase in leaf area in these lines depended on leaf position and growth conditions and that all five lines affected leaf size differently; however, in all cases, an increase in cell number was, entirely or predominantly, responsible for the leaf size enlargement. By analyzing hormone levels, transcriptome, and metabolome, we provide deeper insight into the molecular basis of the growth phenotype for the individual lines. A comparative analysis between these data sets indicates that enhanced organ growth is governed by different, seemingly independent pathways. The analysis of transgenic lines simultaneously overexpressing two growth-enhancing genes further supports the concept that multiple pathways independently converge on organ size control in Arabidopsis.

Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells.

While plasma membrane cholesterol-rich microdomains play a role in cholesterol trafficking, little is known about the appearance and dynamics of cholesterol through these domains in living cells. The fluorescent cholesterol analog 6-dansyl-cholestanol (DChol), its biochemical fractionation, and confocal imaging of L-cell fibroblasts contributed the following new insights: i) fluorescence properties of DChol were sensitive to microenvironment polarity and mobility; (ii) DChol taken up by L-cell fibroblasts was distributed similarly as cholesterol and preferentially into cholesterol-rich vs. -poor microdomains resolved by affinity chromatography of purified plasma membranes; iii) DChol reported similar polarity (dielectric constant near 18) but higher mobility near phospholipid polar head group region for cholesterol in purified cholesterol-rich versus -poor microdomains; and iv) real-time confocal imaging, quantitative colocalization analysis, and fluorescence resonance energy transfer with cholesterol-rich and -poor microdomain markers confirmed that DChol preferentially localized in plasma membrane cholesterol-rich microdomains of living cells. Thus, DChol sensed a unique, relatively more mobile microenvironment for cholesterol in plasma membrane cholesterol-rich microdomains, consistent with the known, more rapid exchange dynamics of cholesterol from cholesterol-rich than -poor microdomains.

Effect of dietary fiber on azoxymethane-induced intestinal carcinogenesis in rats.

The effect of alfalfa, bran, and cellulose on intestinal tumor formation and fecal billary steroid levels was studied in male Sprague-Dawley rats given injections of azoxymethane (AOM). Animals received weekly injections of 8 mg AOM/kg and were fed diets containing 10% fiber (wt/wt) and 35% beef fat or 20 or 30% fiber and about 6% beef fat. Control animals in each instance were fed fiber-free diets. The addition of 10% fiber to the high-fat diet did not significantly reduce the intestinal tumor frequency (average No. of tumors/rat). However, addition of 20 or 30% fiber to the 6% fat diet significantly reduced the intestinal tumor frequency. The concentration of fecal biliary steroids (mg/g dry feces) was significantly lowered in the groups with reduced tumor frequencies, whereas the total excretion of fecal biliary steroids (mg/day) did not show a similar correlation. These observations suggest that intestinal tumor frequency can be reduced by increased dietary fiber only when fat intake is not at a high level. The effect of fiber may be due to dilution of promoters and/or carcinogens in the intestinal tract.

The aminosterol antibiotic squalamine permeabilizes large unilamellar phospholipid vesicles.

The ability of the shark antimicrobial aminosterol squalamine to induce the leakage of polar fluorescent dyes from large unilamellar phospholipid vesicles (LUVs) has been measured. Micromolar squalamine causes leakage of carboxyfluorescein (CF) from vesicles prepared from the anionic phospholipids phosphatidylglycerol (PG), phosphatidylserine (PS), and cardiolipin. Binding analyses based on the leakage data show that squalamine has its highest affinity to phosphatidylglycerol membranes, followed by phosphatidylserine and cardiolipin membranes. Squalamine will also induce the leakage of CF from phosphatidylcholine (PC) LUVs at low phospholipid concentrations. At high phospholipid concentrations, the leakage of CF from PC LUVs deviates from a simple dose-response relationship, and it appears that some of the squalamine can no longer cause leakage. Fluorescent dye leakage generated by squalamine is graded, suggesting the formation of a discrete membrane pore rather than a generalized disruption of vesicular membranes. By using fluorescently labeled dextrans of different molecular weight, material with molecular weight /=10,000 is retained. Negative stain electron microscopy of squalamine-treated LUVs shows that squalamine decreases the average vesicular size in a concentration-dependent manner. Squalamine decreases the size of vesicles containing anionic phospholipid at a lower squalamine/lipid molar ratio than pure PC LUVs. In a centrifugation assay, squalamine solubilizes phospholipid, but only at significantly higher squalamine/phospholipid ratios than required for either dye leakage or vesicle size reduction. Squalamine solubilizes PC at lower squalamine/phospholipid ratios than PG. We suggest that squalamine complexes with phospholipid to form a discrete structure within the bilayers of LUVs, resulting in the transient leakage of small encapsulated molecules. At higher squalamine/phospholipid ratios, these structures release from the bilayers and aggregate to form either new vesicles or squalamine/phospholipid mixed micelles.

Squalamine is not a proton ionophore.

Squalamine, an aminosterol antibiotic isolated from the dogfish shark, creates relatively large defects in phospholipid bilayers, allowing the unrestricted translocation of small molecules across these compromised membranes (B.S. Selinsky, Z. Zhou, K.G. Fotjik, S. R. Jones, N.R. Dollahon, A.E. Shinnar, Biochim. Biophys. Acta 1370 (1998) 218-234). However, an aminosterol structurally similar to squalamine was found to act as a proton ionophore in anionic phospholipid vesicles. In contrast with squalamine, gross membrane disruption was not observed with this synthetic analog (G. Deng, T. Dewa, S.L. Regen, J. Am. Chem. Soc. 118 (1996) 8975-8976). In this report, the ionophoric activity of squalamine was tested in anionic and zwitterionic phospholipid vesicles. No ionophoric activity was observed for squalamine in vesicles comprised of phosphatidylglycerol (PG), phosphatidylcholine (PC), or a mixture of the two lipids. Experiments using radiolabeled squalamine indicated that all of the squalamine added to PG vesicles remained with the vesicles, while approximately one-half of the squalamine added to PC vesicles was incorporated. We have synthesized the aminosterol analog of squalamine possessing ionophoric activity, and its ionophoric activity in PG vesicles was confirmed. The synthetic compound possessed no measurable lytic activity when added to preformed phospholipid vesicles. As both compounds possess significant antimicrobial activity, these results suggest that either multiple mechanisms for the antimicrobial activity of aminosterols exist, depending upon the aminosterol structure, or possibly an unrelated common mechanism for antimicrobial activity remains to be discovered.

Interaction of the polyene antibiotic filipin with model and natural membranes containing plant sterols.

The interaction of the polyene antibiotic, filipin, with individual or mixed plant sterols (stigmasterol, sitosterol, campesterol and 24-methylpollinastanol) incorporated into large unilamellar vesicles (LUV) of soybean phosphatidylcholine (PC) as well as the filipin interaction with purified membrane fractions from maize roots containing these sterols was investigated by ultraviolet (UV) absorption and and circular dichroism (CD) spectroscopy. With both types of membrane preparation, dramatic changes in the UV absorption and CD spectra of the antibiotic were evidenced. When LUV containing stigmasterol, sitosterol and/or campesterol were incubated with low filipin concentrations (i.e., for filipin/sterol molar ratios (rst) lower than 1), CD signal characteristic of the formation of filipin-sterol complexes were observed. At higher rst values, the filipin-sterol interaction was shown to be in competition with a filipin-phospholipid interaction. With 24-methylpollinastanol-containing LUV, the filipin-phospholipid interaction was detected even at rst values lower than 1, which suggests a lower affinity of filipin for this sterol and emphasizes the structural differences between delta 5-sterols and 9 beta,19-cyclopropylsterols. With sterol-free soybean PC LUV, a filipin-phospholipid interaction could also be evidenced. With maize root cell membranes containing either delta 5-sterols or 9 beta,19-cyclopropylsterols, CD spectra similar to those obtained in the presence of LUV having these sterols as components were observed. Thus, the protein component of the membranes does not appear to be an important feature.

The gene for a xyloglucan endotransglucosylase/hydrolase from Cicer arietinum is strongly expressed in elongating tissues.

We have isolated a Cicer arietinum cDNA clone (CaXTH1) encoding a protein that belongs to the family 16 of glycosyl hydrolases and has all the conserved features of xyloglucan endotransglucosylase/hydrolases (XTH) proteins, including the presence of a highly conserved domain (DEIDFEFLG) and four Cys which suggest the potential for forming disulfide bonds. These facts indicate that CaXTH1 encodes a putative XTH. This chickpea protein showed a high level of sequence identity with group 1 XTHs that have xyloglucan endotransglucosylase (XET) activity. CaXTH1 was selected by differential screening of a cDNA library constructed using mRNA from C. arietinum polyethylene glycol (PEG) treated epicotyls, as a clone whose expression decreased when epicotyl growth was inhibited by PEG. CaXTH1 shows an expression pattern that seems to be specific for growing tissue, mostly epicotyls and the growing internodes of adult stems. CaXTH1 mRNA was not detected in any other organs of either seedlings or adult plants. CaXTH1 mRNA was abundant when epicotyls are actively growing; there was almost no expression after PEG-treatment. CaXTH1 was up-regulated by indole acetic acid (IAA) and brassinolides (BR), showing the highest transcript levels after IAA plus BR treatment. In situ hybridization study revealed that CaXTH1 is mainly expressed in epidermal cells, the target of the cell expansion process, and also in vascular tissues. The present results suggest an involvement of the putative XTH encoded by CaXTH1 in the chickpea cell expansion process.

Transfer of steroids and alpha-tocopherol between liposomal membranes.

The transfer of various steroids and alpha-tocopherol between liposomes was examined. The transfer rate of cholest-4-en-3-one and epicholesterol between egg yolk phosphatidylcholine liposomes was much higher than that of cholesterol. Rapid transfer was also noted for coprostanol and 5-cholesten-3 beta-ol-7-one, while the transfer of cholestanol, which has a 3 beta-hydroxyl residue and a planar nucleus structure, was similar to that of cholesterol. These results suggest that the mode of interaction between steroid and phospholipid may be an important factor controlling the rate of transfer. It was found that alpha-tocopherol could also be transferred between liposomes. The transfer rate of alpha-tocopherol was dependent on the temperature and the phospholipid composition.

Hydrophilic 7 beta-hydroxy bile acids, lovastatin, and cholestyramine are ineffective in the treatment of cerebrotendinous xanthomatosis.

We compared the effect of treatments with hydrophilic bile acids (ursodeoxycholic and ursocholic acids), cholestyramine, and lovastatin versus chenodeoxycholic acid in 4 patients with cerebrotendinous xanthomatosis (CTX). Bile acids and bile alcohols in plasma, bile, and urine before and after treatment were quantitated by gas-liquid chromatography. Untreated, all patients showed abnormal biliary bile acid composition: cholic acid (72.7%) and chenodeoxycholic acid (6.2%), and polyhydroxylated C(27)-bile alcohols (10.0%), and elevated plasma cholestanol levels. Treatment with hydrophobic chenodeoxycholic acid inhibited abnormal bile acid synthesis (virtual disappearance of C(27)-bile alcohols from plasma, bile, and urine and marked reduction of plasma cholestanol levels). Hydrophilic ursodeoxycholic and ursocholic acids did not inhibit abnormal bile acid synthesis, while cholestyramine increased abnormal bile acid synthesis (continued increased formation of polyhydroxylated C(27)-bile alcohols and further elevation of plasma cholestanol levels). Lovastatin did not affect abnormal bile acid synthesis or reduce plasma cholestanol levels. The results demonstrate that impaired side-chain oxidation in bile acid synthesis due to mutations of Cyp27 results in increased formation of polyhydroxylated C(27)-bile alcohols and cholestanol in CTX. Hydrophobic chenodeoxycholic acid, but not cholestyramine, lovastatin, or hydrophilic 7beta-hydroxy acids, inhibited the abnormal synthetic pathway. The role of chenodeoxycholic acid in downregulating abnormal bile acid synthesis in CTX is emphasized.

Detection of carriers of cerebrotendinous xanthomatosis.

Patients suffering from cerebrotendinous xanthomatosis (an autosomal recessive inborn error of metabolism) can easily be distinguished from patients not suffering from this disease, as the first excrete large amounts of the bile alcohol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol, in urine, whereas the second do not. In order to find out, whether carriers of cerebrotendinous xanthomatosis can be detected in a biochemical way, we compared known carriers with controls. The urinary excretions of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol of both groups were practically absent and no selection of carriers with cerebrotendinous xanthomatosis could be made on that basis. When, however, carriers and non-carriers were subjected to cholestyramine treatment, by which endogenous bile acid synthesis was stimulated, the urinary excretion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol in the carrier rose considerably, whereas this excretion remained essentially the same in the non-carriers. This test can be of value in the genetic counseling of carriers with cerebrotendinous xanthomatosis and helpful in the detection of newborn patients with cerebrotendinous xanthomatosis.

Effects of cholestanol feeding and cholestyramine treatment on the tissue sterols in the rabbit.

Rabbits were fed diets enriched with cholestanol or cholesterol (3.5 g/wk) for 4-12 weeks. During cholestanol feeding, the concentration of cholestanol in blood serum, liver, heart and aorta increased 15-30 times. In serum and liver, the concentration of cholesterol also increased. Cholestanol-fed rabbits developed inflammatory changes in the liver, with proliferation of small bile ducts. Liver tests were only slightly abnormal. Morphological atherosclerosis of the aorta was only occasionally seen in rabbits receiving cholestanol for eight weeks or less. During cholesterol feeding, the amounts of cholesterol in different tissues increased dramatically, most in the aorta. Morphological atherosclerosis in the aorta was found in all rabbits fed cholesterol-enriched diets for more than four weeks. Brain cholestanol was doubled in rabbits fed cholestanol for eight weeks, whereas brain sterols did not change significantly during cholesterol feeding. After an additional regression period with cholestyramine for eight weeks, the increased content of cholestanol in the brain was unchanged in cholestanol-fed rabbits. These observations are discussed in relation to the cholestanolosis of the brain that develops in the rare inherited human disease cerebrotendinous xanthomatosis.

Preparation of styrene-co-4-vinylpyridine magnetic polymer beads by microwave irradiation for analysis of trace 24-epibrassinolide in plant samples using high performance liquid chromatography.

In the study, a kind of novel styrene-co-4-vinylpyridine (St-co-4-VP) porous magnetic polymer beads was prepared by microwave irradiation using suspension polymerization. Microwave heating preparation greatly reduced the polymerization time to 1h. Physical characteristic tests suggested that these beads were cross-linking and possessed spherical shape, good magnetic response and porous morphologies with a narrow diameter distribution of 70-180 µm. Therefore, these beads displayed the long-term stability after undergoing 100-time extractions. Then, an analytical method for the determination of trace 24-epiBR in plant samples was developed by magnetic polymer bead extraction coupled with high performance liquid chromatography-fluorescence detection. St-co-4-VP magnetic polymer beads demonstrated the higher extraction selectivity for 24-epiBR than other reference compounds. Linear range was 10.00-100.0 µg/L with a relative standard deviation (RSD) of 6.7%, and the detection limit was 6.5 µg/kg. This analytical method was successfully applied to analyze the trace 24-epiBR in cole and breaking-wall rape pollen samples with recoveries of 77.2-90.0% and 72.3-83.4%, respectively, and RSDs were less than 4.1%. The amount of 24-epiBR in real breaking-wall rape pollen samples was found to be 26.2 µg/kg finally. This work proposed a sensitive, rapid, reliable and convenient analytical method for the determination of trace brassinosteroids in complicated plant samples by the use of St-co-4-VP magnetic polymer bead extraction coupled with chromatographic method.

Increased plasma cholestanol and 5 alpha-saturated plant sterol derivatives in subjects with sitosterolemia and xanthomatosis.

We have measured plasma sterol composition in 14 subjects with sitosterolemia and xanthomatosis. In addition to elevated plasma phytosterol (campesterol 16 +/- 7 mg/dl and sitosterol 35 +/- 16 mg/dl) and normal to moderately high cholesterol levels (258 +/- 96 mg/dl), concentrations of 5 alpha-saturated stanols, cholestanol, 5 alpha-campestanol, and 5 alpha-sitostanol were at least 10 times greater than controls. Diets contained plentiful quantities of cholesterol and plant sterols, but only trace amounts of cholestanol (less than 2 mg/day) and no detectable 5 alpha-campestanol and 5 alpha-sitostanol, which indicated that the 5 alpha-saturated stanols were formed endogenously. Treatment with cholestyramine reduced plasma cholesterol and phytosterol levels by 45% and 5 alpha-saturated stanols by 55%. These results indicate that abnormally high plasma concentrations of cholestanol, 5 alpha-campestanol, and 5 alpha-sitostanol are found in subjects with sitosterolemia and xanthomatosis, and that treatment with cholestyramine effectively reduced elevated plasma sterol levels.

Aquaporin isoforms responsive to salt and water stresses and phytohormones in radish seedlings.

Aquaporins in the plasma and vacuolar membranes play a key role in the intercellular and intracellular water transport in plants. First, we quantitated the absolute amounts for mRNAs of eight aquaporin isoforms in hypocotyls of radish seedlings. Then, we investigated the effects of salt and water stresses (150 mM NaCl, 300 mM mannitol and 20% polyethylene glycol) and phytohormones (gibberellic acid, abscisic acid and brassinolide) on the mRNA and protein levels of aquaporins in the plasma membrane (RsPIP1-1, 1-2, 1-3, 2-1, 2-2 and 2-3) and vacuolar membrane (RsTIP1-1 and 2-1). The mRNA and protein levels of RsTIP1-1, RsTIP2-1, RsPIP1-1, RsPIP1-2 and RsPIP1-3 were comparatively constant. In contrast, mannitol treatment altered the mRNA levels of RsPIP2-1, RsPIP2-2 and RsPIP2-3 in roots. Immunoblot analysis showed that the RsPIP2-1 protein level was increased by NaCl treatment and decreased by treatment with mannitol and polyethylene glycol. Gibberellic acid and abscisic acid suppressed the levels of mRNAs of RsPIP2-1, RsPIP2-2 and RsPIP2-3 and the protein level of RsPIP2-1 in roots. On the other hand, the protein levels of RsPIP1-group members and RsTIPs were scarcely changed by these phytohormones. In the case of hypocotyls and cotyledons, the mRNA and protein levels of eight isoforms were not markedly affected by any treatment. These results indicate that aquaporins in the root, especially the RsPIP2 group, may be a stress responsive type of aquaporin at least in the protein level.