A comparative study of the effects of cholesterol and sclareol, a bioactive labdane type diterpene, on phospholipid bilayers

Sclareol (labd-14-ene-8,13-diol) is a highly water-insoluble molecule that belongs to the labdane type diterpenes and is characterized as a biologically active molecule, due to its cytotoxic and cytostatic effects against human leukemic cell lines. A superimposition study between sclareol and cholesterol, based on their corresponding hydrophobic and polar molecular segments calculated from their lipophilic profiles, revealed their spatial similarities. This structural similarity between the two molecules prompted us to compare their effects on the structure and stability of phospholipid dipalmitoylphosphatidylcholine (DPPC) membranes. Differential scanning calorimetry (DSC) was applied to compare the thermal changes caused by either cholesterol or sclareol when are incorporated in DPPC bilayers. The results showed that sclareol is incorporated into phospholipid model membranes and mimics the thermal effects of cholesterol especially at concentrations up to X(sclareol)=9.1 mol%. These effects can be summarized as the abolition of pre-transition, lowering of the main phase transition and reduction of the enthalpy change (DeltaH) of the gel to liquid-crystalline phase transition of DPPC bilayers. At concentrations X> or =16.7 mol%, sclareol and cholesterol caused different heterogeneity in lipid bilayers or a reversible transition from a vesicular suspension to an extended peak bilayer network. This different fluidization, exerted by the two molecules at high concentration, may be related to their different stability and the z-average mean diameter of the liposomes they form. Small unilamellar vesicles, prepared by the thin film hydration method showed that DPPC bilayers containing a high concentration of sclareol in equimolar ratio sclareol:cholesterol were unstable, in contrast to the ones containing only cholesterol.     

Effect of the nature of the 3beta-substitution in manoyl oxides on the thermotropic behavior of DPPC lipid bilayer and on DPPC liposomes

Functionalized manoyl oxide derivatives have been proved over the years to evoke several biological responses. Among them, 3beta-hydroxy-manoyl oxide (1) and 3beta-acetoxy-manoyl oxide (2) have been shown to exhibit in vitro antimicrobial and cytotoxic activity, while N-imidazole-3 beta-thiocarbonyl ester of manoyl oxide (3) was found to exhibit potent cytotoxic effect. Their partitioning into phospholipid bilayers may lead to membrane structure modifications that are crucial in liposome development as they may influence their maintenance and integrity. DSC was used to study the modifications induced in DPPC bilayers by incorporating increasing concentrations of the three manoyl oxide derivatives. All derivatives were found to strongly affect the bilayer structural organization in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase and the induction of a lateral phase separation in clustering domains. Derivatives 1 and 3 were incorporated into DPPC liposomes and their physicochemical stability was monitored at 4 degrees C. The stability of liposomes was strongly influenced by the presence of 1 and 3 at any molar ratio studied. DPPC/1 liposomes were found to retain its stability for 48 h at low concentration of 10% mol, while at higher concentrations up to 30% mol they collapsed into aggregated material. In all cases DPPC/3 liposomes were found unstable and sticky aggregated structures precipitated from the bulk suspension.     

Labdane-type diterpenes: thermal effects on phospholipid bilayers, incorporation into liposomes and biological activity

Labd-13(E)-ene-8alpha,15-diol (1) and its derivative labd-13(E)-ene-8alpha-ol-15-yl-acetate (2) are water insoluble biological active molecules and their structures were elucidated using NMR and X-ray techniques. Differential scanning calorimetry (DSC) was applied to study the thermal effects of 1 and 2 on DPPC bilayers. Liposomes composed of egg phosphatidylcholine/dipalmytoylphosphatidylglycerol (9:0.1 molar ratio) were prepared by the thin-film hydration method and were used for incorporating 1 and 2. Free and liposomal 1 and 2 were tested for their activity against human cancer cell lines using the sulphorhodamine B assay. The effect of 1 and 2 on DPPC bilayers caused abolition of the pre-transition temperature, lowering of the main phase transition and reduction of the transition enthalpy only in the presence of cholesterol. The liposomes that have been designed and developed offer high incorporation efficiency; 62.4% (0.369 drug/lipid molar ratio) and 99.7% (0.661 drug/lipid molar ratio) for 1 and 2, respectively. Liposomal 2 showed growth-inhibiting activity against the majority of the tested cell lines.     

Effect of the Nature of the 3β-Substitution in Manoyl Oxides on the Thermotropic Behavior of DPPC Lipid Bilayer and on DPPC Liposomes

Functionalized manoyl oxide derivatives have been proved over the years to evoke several biological responses. Among them, 3β-hydroxy-manoyl oxide (1) and 3β-acetoxy-manoyl oxide (2) have been shown to exhibit in vitro antimicrobial and cytotoxic activity, while N-imidazole-3 β-thiocarbonyl ester of manoyl oxide (3) was found to exhibit potent cytotoxic effect. Their partitioning into phospholipid bilayers may lead to membrane structure modifications that are crucial in liposome development as they may influence their maintenance and integrity. DSC was used to study the modifications induced in DPPC bilayers by incorporating increasing concentrations of the three manoyl oxide derivatives. All derivatives were found to strongly affect the bilayer structural organization in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase and the induction of a lateral phase separation in clustering domains. Derivatives 1 and 3 were incorporated into DPPC liposomes and their physicochemical stability was monitored at 4°C. The stability of liposomes was strongly influenced by the presence of 1 and 3 at any molar ratio studied. DPPC/1 liposomes were found to retain its stability for 48 h at low concentration of 10% mol, while at higher concentrations up to 30% mol they collapsed into aggregated material. In all cases DPPC/3 liposomes were found unstable and sticky aggregated structures precipitated from the bulk suspension.     

HLA class II allele frequencies in the Lebanese population

Aims

Being one of the most polymorphic genetic systems , the Human Leukocyte Antigen system is divided into class I (HLA-A, HLA-B and HLA-C) and class II (HLA-DP, -DQ and -DR). This study is the first and largest of its kind to describe the distribution of HLA-DQB1 and HLA-DRB1 alleles in Lebanon and the region.

Methods

Respectively, 560 and 563 Lebanese individuals referred for HLA typing and possible bone marrow/kidney donation were tested for HLA-DQB1 and HLA-DRB1 alleles using the polymerase chain reaction/sequence specific priming (PCR-SSP) method.

Results

Our data were compared to that of several populations with interesting common findings between the Lebanese, Jordanian, Bahraini, Saudi, Kuwaiti, Tunisian, Korean, Japanese, Thai, Irish, Bulgarian and Polish populations.

Conclusion

These data about the Lebanese population are going to aid future researchers to study the relation of HLA-DQB1 and HLA-DRB1 alleles with major and common diseases in the Lebanese population and will add to the available international literature associated with these loci. In addition it will serve as a reference for the future national bone marrow registry program in our country. We also reviewed the literature for the described association between HLA-DRB1 and -DQB1 loci and different disease entities.     

Conservation of key members in the course of the evolution of the insulin signaling pathway

Our understanding of the evolution of the insulin signaling pathway (ISP) is still incomplete. One intriguing unanswered question is the explanation of the emergence of the glucostatic role of insulin in mammals. To find out whether this is due to the development of new sets of signaling transduction elements in these organisms, or to the establishment of new interactions between pre-existing proteins, we rebuilt putative orthologous ISPs in 17 eukaryotic organisms. Then, we computed the conservation of orthologous ISPs at different levels, from sequence similarity of orthologous proteins to co-evolution of interacting domains. We found that the emergence of glucostatic role in mammals can neither be explained by the development of new sets of signaling elements, nor by the establishment of new interactions between pre-existing proteins. The comparison of orthologous IRS molecules indicates that only in mammals have they acquired their complete functionality as efficient recruiters of effector sub-pathways.     

ProtIdent: a web server for identifying proteases and their types by fusing functional domain and sequential evolution information

Proteases are vitally important to life cycles and have become a main target in drug development. According to their action mechanisms, proteases are classified into six types: (1) aspartic, (2) cysteine, (3) glutamic, (4) metallo, (5) serine, and (6) threonine. Given the sequence of an uncharacterized protein, can we identify whether it is a protease or non-protease? If it is, what type does it belong to? To address these problems, a 2-layer predictor, called "ProtIdent", is developed by fusing the functional domain and sequential evolution information: the first layer is for identifying the query protein as protease or non-protease; if it is a protease, the process will automatically go to the second layer to further identify it among the six types. The overall success rates in both cases by rigorous cross-validation tests were higher than 92%. ProtIdent is freely accessible to the public as a web server at http://www.csbio.sjtu.edu.cn/bioinf/Protease.     

Sclareol induces apoptosis in human HCT116 colon cancer cells <Emphasis Type="Italic">in vitro</Emphasis> and suppression of HCT116 tumor growth in immunodeficient mice

Labd-14-ene-8, 13-diol (sclareol) is a labdane-type diterpene, which has demonstrated significant cytotoxic activity against human leukemic cell lines, but its effect on solid tumor-derived cells is uknown. Here, we demonstrate that addition of sclareol to cultures of human colon cancer HCT116 cells results in inhibition of DNA synthesis, arrest of cells at the G₁ phase of the cell cycle, activation of caspases-8, -9, PARP degradation, and DNA fragmentation, events characteristic of induction of apoptosis. Intraperitoneal (ip) administration of sclareol alone, at the maximum tolerated dose, was unable to induce suppression of growth of HCT116 tumors established as xenografts in immunodeficient SCID mice. In contrast, ip administration of liposome-encapsulated sclareol, following a specific schedule, induced suppression of tumor growth by arresting tumor cell proliferation as assessed by detecting the presence of the cell proliferation-associated nuclear protein, Ki67, in thin tumor sections. These findings suggest that sclareol incorporated into liposomes may possess chemotherapeutic potential for the treatment of colorectal and other types of human cancer.     

The behaviors of Ca(2+)-ATPase embedded in interdigitated bilayer.

We investigated the behavior of a membrane protein, Ca(2+)-ATPase, in interdigitated phospholipid bilayers. The results showed that Ca(2+)-ATPase does not cause significant alterations in the interdigitation of 16:0 LPC/DPPC (27.0 mol% LPC) vesicles when it is reconstituted with lipids. Intrinsic fluorescence, acrylodan fluorescent adducts, and CD spectra indicated that Ca(2+)-ATPase, when embedded in interdigitated bilayer structures, is more exposed to the hydrophilic environment and has a looser structure than when embedded in non-interdigitated bilayers. The interdigitation of acyl chains induces a rapid loss of enzyme activity. It is suggested that interdigitated bilayer structures may play an important role as negative regulatory factors in physiological functions.     

Molecular insights into NF2/Merlin tumor suppressor function

The FERM domain protein Merlin, encoded by the NF2 tumor suppressor gene, regulates cell proliferation in response to adhesive signaling. The growth inhibitory function of Merlin is induced by intercellular adhesion and inactivated by joint integrin/receptor tyrosine kinase signaling. Merlin contributes to the formation of cell junctions in polarized tissues, activates anti-mitogenic signaling at tight-junctions, and inhibits oncogenic gene expression. Thus, inactivation of Merlin causes uncontrolled mitogenic signaling and tumorigenesis. Merlin’s predominant tumor suppressive functions are attributable to its control of oncogenic gene expression through regulation of Hippo signaling. Notably, Merlin translocates to the nucleus where it directly inhibits the CRL4^DCAF1 E3 ubiquitin ligase, thereby suppressing inhibition of the Lats kinases. A dichotomy in NF2 function has emerged whereby Merlin acts at the cell cortex to organize cell junctions and propagate anti-mitogenic signaling, whereas it inhibits oncogenic gene expression through the inhibition of CRL4^DCAF1 and activation of Hippo signaling. The biochemical events underlying Merlin’s normal function and tumor suppressive activity will be discussed in this Review, with emphasis on recent discoveries that have greatly influenced our understanding of Merlin biology.     

The effect of cholesterol in a liposomal Muc1 vaccine

A liposomal Muc1 mucin vaccine for treatment of adenocarcinomas was formulated by incorporating a synthetic Muc1 mucin-based lipopeptide and Lipid A into a DPPC/cholesterol bilayer. Vaccination of mice with the liposomal formulation produced a peptide-specific immune response dependent on the cholesterol content. The response occurred at a threshold of 20-23 mol% cholesterol, and was optimal at cholesterol levels of > or =30 mol%. To understand this cholesterol dependency, we studied the effect of cholesterol on the liposomal bilayer and surface properties. Freeze-fracture electron microscopy showed a unique surface texture that was codependent upon cholesterol (> or =20 mol%) and lipopeptide content. Fluorescence anisotropy measurements exhibited a significant decrease in the rotational motion of 1,6-diphenyl-1,3,5-hexatriene in formulations containing >20 mol% cholesterol and only in the presence of the lipopeptide. At 20 mol% cholesterol and with lipopeptide, DSC showed a significant increase in the main phase transition of the DPPC bilayers, while Raman spectroscopy indicated a more ordered arrangement of DPPC molecules compared to control liposomes containing DPPC/cholesterol alone. Taken together, the data suggest the presence of lipopeptide-rich microdomains at and above a threshold of 20 mol% cholesterol that may play a role in the induction of a peptide-specific immunological response.     

Effect of lysophosphatidylcholine on behavior and structure of phosphatidylcholine liposomes

Differential scanning calorimetry (DSC), fluorescence polarization and X-ray diffraction were per-formed to investigate the kinetics of the micellar to the lamellar phase transition of dipalmitoylphosphatidylcholine/1-palmitoylphosphatidylcholine (16:0 LPC/DPPC) liposomes at gel phase. With a 16:0 LPC concentration up to 27 mol% only the sharp main transition with relatively high enthalpy (△H) values of DPPC was observed. Increasing 16 : 0 LPC concentration, the phase transition was broadened and the transition enthalpy was decreased and finally totally disappeared. The fluorescence probes of 3AS, 9AS, 12AS, and 16AP were employed, respectively, to detect the mo-bility of various sites of carbon chains of DPPC or 16:0 LPC/DPPC liposomes. It was shown that DPPC liposomes formed in the absence of 16:0 LPC always had a fluidity gradient in both gel and liquid-crystalline phase, while in the presence of 14.1 mol% and 27.0 mol% 16:0 LPC in the mixtures, the fluidity gradient tended to disappear below 40℃:     

Thiocyanate and bromide ions influence the bilayer structural parameters of phosphatidylcholine bilayers

The influence of monovalent cations and anions on the structural parameters of dipalmitoylphosphatidylcholine (DPPC) bilayers was examined at 25 degrees C using X-ray diffraction. It was shown that monovalent salts, in general, have little effect on lipid packing within the bilayer. However, fully hydrated DPPC bilayers in 1 M KSCN pack in an interdigitated acyl chain phase. This is the first observation of an ion-induced interdigitated bilayer phase in a zwitterionic lipid. In addition, gel state DPPC bilayers in 1 M KBr imbibe approx. 10 A more solvent than bilayers in water. The influence of these same salts on the phase transitions of DPPC bilayers was also examined using high-resolution differential scanning calorimetry. These results are discussed in terms of ion-induced changes in solvent and solvent/bilayer structure.     

Comparative calorimetric and spectroscopic studies of the effects of cholesterol and epicholesterol on the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayer membranes

We carried out comparative differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol (Chol) and epicholesterol (EChol) on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine (DPPC) bilayers. EChol is an epimer of Chol in which the axially oriented hydroxyl group of C3 of Chol is replaced by an equatorially oriented hydroxyl group, resulting in a different orientation of the hydroxyl group relative to sterol fused ring system. Our calorimetric studies indicate that the incorporation of EChol is more effective than Chol is in reducing the enthalpy of the pretransition of DPPC. EChol is also initially more effective than Chol in reducing the enthalpies of both the sharp and broad components of the main phase transition of DPPC. However, at higher EChol concentrations (~ 30-50 mol%), EChol becomes less effective than Chol in reducing the enthalpy and cooperativity of the main phase transition, such that at sterol concentrations of 50 mol%, EChol does not completely abolish the cooperative hydrocarbon chain-melting phase transition of DPPC, while Chol does. However, EChol does not appear to form a calorimetrically detectable crystallite phase at higher sterol concentrations, suggesting that EChol, unlike Chol, may form dimers or lower order aggregates at higher sterol concentrations. Our spectroscopic studies demonstrate that EChol incorporation produces more ordered gel and comparably ordered liquid-crystalline bilayers compared to Chol, which are characterized by increased hydrogen bonding in the glycerol backbone region of the DPPC bilayer. These and other results indicate that monomeric EChol is less miscible in DPPC bilayers than is Chol at higher sterol concentrations, but perturbs their organization to a greater extent at lower sterol concentrations, probably due primarily to the larger effective cross-sectional area of the EChol molecule. Nevertheless, EChol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers.     

Triterpenoids of Salvia horminum,constitution of a new diol

Lupeol, lup-(20)29-ene-2α,3β-diol and a new triterpenic alcohol olean-(13)18-ene-2β,3β-diol were isolated from the petrol extract of air dried Salvia horminum and their structures were determined.     

Proposal of Ensifer psoraleae sp. nov., Ensifer sesbaniae sp. nov., Ensifer morelense comb. nov. and Ensifer americanum comb. nov

In a survey of rhizobia associated with the native legumes in Yunnan Province, China, seven and nine strains isolated from the root nodules of Psoralea corylifolia, Sesbania cannabina and Medicago lupulina were respectively classified into the novel genomic species groups I and II in the genus Ensifer (former Sinorhizobium) based on the sequence analyses of the 16S rRNA gene. Analyses of concatenated housekeeping genes (atpD, recA and glnII) further revealed that they were distinct lineages in the genus, and group I was most similar to Ensifer terangae and Ensifer garamanticus (both with 94.2% similarity), while group II was most similar to Ensifer adhaerens (94.0%). These groups could be distinguished from closely related species by DNA–DNA relatedness, MALID-TOF MS, cellular fatty acid profiles and a series of phenotypic characters. Therefore, two novel species were proposed: Ensifer psoraleae sp. nov. (seven strains, type strain CCBAU 65732^T = LMG 26835^T = HAMBI 3286^T) and Ensifer sesbaniae sp. nov. (nine strains, type strain CCBAU 65729^T = LMG 26833^T = HAMBI 3287^T). They had a DNA G + C mol% (T_m) of 58.9 and 60.4, respectively. Both of the type strains formed effective nodules on common bean (Phaseolus vulgaris) and their hosts of origin. In addition, the previously described species Sinorhizobium morelense and Sinorhizobium americanum were renamed as Ensifer morelense comb. nov. and Ensifer americanum comb. nov. according to the accumulated data from different studies.     

Labdane diterpenes from Cistus symphytifolius

The investigation of the aerial part of Cistus symphytifolius afforded, in addition to sitosterol, trimethoxykaempferol, cativic acid, labdenic acid, labdanolic acid and labdan-8α,15-diol, three new bicyclic diterpenes: cistadienic acid, cistenolic acid and labd-13(E)- ene-8α,15-diol. The structures of these were determined by spectral studies and correlations. CD spectral studies showed that cistenolic acid and salvic acid are enantiomeric compounds, so the stereochemistry of salvic acid (7α-hydroxy-labd-8(17)-ene-15-oic acid) should be changed to 7β-hydroxy-eperu-8(17)-ene-15-oic acid (7β-hydroxy-ent-labd-8(17)-ene-15-oic acid).     

Genotypic and phenotypic characterization of Brazilian patients with GM1 gangliosidosis

GM1 gangliosidosis is a lysosomal disorder caused by β-galactosidase deficiency due to mutations in the GLB1 gene. It is a rare neurodegenerative disorder with an incidence of about 1:100,000–1:200,000 live births worldwide. Here we review GLB1 mutations and clinical features from 65 Brazilian GM1 gangliosidosis patients. Molecular analysis showed 17 different mutations and c.1622–1627insG was the most frequent, accounting for 50% of the alleles. Cognitive impairment was the main clinical sign, observed in 82% of patients, followed by hepatosplenomegaly observed in 56% of patients. It was possible to establish a significant correlation between age at onset of symptoms preceding the first year of life and the presence of the mutation c.1622–1627insG (p = 0.03). Overall our findings differ from literature and represent the exclusive genotypic profile found in Brazilian GM1 gangliosidosis patients.     

A calorimetric and spectroscopic comparison of the effects of ergosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed comparative DSC and FTIR spectroscopic measurements of the effects of cholesterol (Chol) and ergosterol (Erg) on the thermotropic phase behavior and organization of DPPC bilayers. Ergosterol is the major sterol in the biological membranes of yeasts, fungi and many protozoa. It differs from Chol in having two additional double bonds, one in the steroid nucleus at C7-8 and another in the alkyl chain at C22-23. Erg also has an additional methyl group in the alkyl chain at C24. Our DSC studies indicate that the incorporation of Erg is more effective than Chol is in reducing the enthalpy of the pretransition. At lower concentrations Erg is also more effective than Chol in reducing the enthalpies of both the sharp and broad components of main phase transition. However, at sterol concentrations from 30 to 50 mol%, Erg is generally less effective at reducing the enthalpy of the broad components and does not completely abolish the cooperative hydrocarbon chain-melting phase transition at 50 mol%, as does Chol. Nevertheless, in this higher ergosterol concentration range, there is no evidence of the formation of ergosterol crystallites. Our FTIR spectroscopic studies demonstrate that Erg incorporation produces a similar ordering of liquid-crystalline DPPC bilayers as does Chol, but an increased degree of hydrogen bonding of the fatty acyl carbonyl groups in the glycerol backbone region of the DPPC bilayer. These and other results indicate that Erg is less miscible in DPPC bilayers at higher concentrations than is Chol. Finally, we provide a tentative molecular explanation for the comparative experimental and computation results obtained for Erg and Chol in phospholipid bilayers, emphasizing the dynamic conformational differences between these two sterols.     

Dimeric progestins from rhizomes of Ligusticum chuanxiong

Five dimeric phthalides were isolated from rhizomes of Ligusticum chuanxiong and their structures deduced based on spectral data. All compounds and their parent extracts were assessed for progesterone-like activity using a progesterone receptor driven reporter-gene bioassay. Among all the compounds, riligustilide, displayed weak progesterone-like activity (EC50 approximately 81 microM), whereas, (3Z')-(3a'R,6'R,3R,6R,7R)-3,8-dihydro-6.6',7.3a'-diligustilide (Mr: 382, EC50 approximately 90 nM), was found to be a potent and specific activator of the progesterone receptor. Levistolide A, although having a very similar plenary structure, was inactive indicating the importance of stereochemistry of chiral centers and flexibility of butylidene side chain for progestogenic activity. These bioactive phthalides and their parent extracts (EC50 approximately 8 microg/ml) may have utility for treatment of conditions requiring progesterone action.