Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b 5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.

Coated pits,endosome CURLing and intracellular receptor--ligand trafficking

In mediating endocytosis of extracellular macromolecules; the major mechanism in which cells ingest nutrients, degrade hormones and maintain the protein and lipid compositions of their organelle membrane, the cell surface receptors encounter 'coated pits', migrate continuously from one organelle to another, deliver the 'cargo' and often recycle back to the cell surface. This article is an attempt to give an account of the recent advances in our understanding of the molecular events involved in the 'round trip itinerary' of cell surface receptors.

Membranes of Cationic Gemini Lipids based on Cholesterol with Hydroxyl Headgroups and their Interactions with DNA and Phospholipid

Two series of cholesterol-based cationic gemini lipids with and without hydroxyl functions at the headgroups possessing different lengths of polymethylene -(CH2)(n)-] (n = 3, 4, 5, 6, 12) spacer have been synthesized. Each gemini lipid formed stable suspension in water. The suspensions of these gemini lipids in water were investigated using transmission electron microscopy, dynamic light scattering, zeta potential measurements and X-ray diffraction to characterize the nature of the individual aggregates formed therein. The aggregation properties of these gemini lipids in water were found to strongly depend upon the length of the spacer and the presence of hydroxyl group at the headgroup region. Lipoplex formation (DNA binding) and the release of the DNA from such lipoplexes were performed to understand the nature of interactions that prevail between these cationic cholesterol aggregates and duplex DNA. The interactions between such gemini lipids and DNA depend both on the presence of OH on the headgroups and the spacer length between the headgroups. Finally, we studied the effect of incorporation of each cationic gemini lipid into dipalmitoyl phosphatidylcholine vesicles using differential scanning calorimetry. The properties of the resulting mixed membranes were found again to depend upon the nature of the headgroup and the spacer chain length.

Intracellular Pathogen Sensor NOD2 Programs Macrophages to Trigger Notch1 Activation

Intracellular pathogen sensor, NOD2, has been implicated in regulation of wide range of anti-inflammatory responses critical during development of a diverse array of inflammatory diseases; however, underlying molecular details are still imprecisely understood. In this study, we demonstrate that NOD2 programs macrophages to trigger Notch1 signaling. Signaling perturbations or genetic approaches suggest signaling integration through cross-talk between Notch1-PI3K during the NOD2-triggered expression of a multitude of immunological parameters including COX-2/PGE(2) and IL-10. NOD2 stimulation enhanced active recruitment of CSL/RBP-Jk on the COX-2 promoter in vivo. Intriguingly, nitric oxide assumes critical importance in NOD2-mediated activation of Notch1 signaling as iNOS(-/-) macrophages exhibited compromised ability to execute NOD2-triggered Notch1 signaling responses. Correlative evidence demonstrates that this mechanism operates in vivo in brain and splenocytes derived from wild type, but not from iNOS(-/-) mice. Importantly, NOD2-driven activation of the Notch1-PI3K signaling axis contributes to its capacity to impart survival of macrophages against TNF-alpha or IFN-gamma-mediated apoptosis and resolution of inflammation. Current investigation identifies Notch1-PI3K as signaling cohorts involved in the NOD2-triggered expression of a battery of genes associated with anti-inflammatory functions. These findings serve as a paradigm to understand the pathogenesis of NOD2-associated inflammatory diseases and clearly pave a way toward development of novel therapeutics.

Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin CDNA

A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)(i)], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)(i)] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells al all stages of development exhibit a sharp transient increase in [(Ca2+)(i)] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)(i)] and show three times as large a response to cAMP stimulation as prespore cells.

Syntheses, Transfection Efficacy and Cell Toxicity Properties of Novel Cholesterol-based Gemini Lipids having Hydroxyethyl Head group

We have synthesized five new cholesterol based gemini cationic lipids possessing hydroxyethyl (-CH2CH2OH) function on each head group, which differ in the length of the polymethylene spacer chain. These gemini lipids are important for gene delivery processes as they possess pre-optimized molecular features, e. g., cholesterol backbone, ether linkage and a variable spacer chain between both the headgroups of the gemini lipids. Cationic liposomes were prepared from each of these lipids individually and as a mixture of individual cationic gemini lipid and 1,2-dioleoyl phosphatidylethanolamine (DOPE). Each gemini lipid based formulation induced better transfection activity than that of their monomeric counterpart. One such gemini lipid with a -(CH2)(12)-spacer, HG-12, showed dramatic increase in the mean fluorescence intensity due to the expression of green-fluorescence protein (GFP) in the presence of 10% FBS compared to the conditions where there was no serum. Other gemini lipids retained their gene transfection efficiency without any marked decrease in the presence of serum. The only exception was seen with the gemini with a -(CH2)(3)-spacer, HG-3, which on gene transfection in the presence of 10% FBS lost similar to 70% of its transfection efficiency. Overall the gemini lipid with a -(CH2)(5)-spacer, HG-5, showed the highest transfection activity at N/P (lipid/DNA) ratio of 0.5 and lipid : DOPE molar ratio of 2. Upon comparison of the relevant parameters, e. g., %-transfected cells, the amount of DNA transfected to each cell and %-cell viability all together against Lipofectamine 2000, one of the best commercial transfecting agents, the optimized lipid formulation based on DOPE/HG-5 was found to be comparable. In terms of its ability to induce gene-transfer in the presence of serum and shelf-life DOPE/HG-5 liposome was found to be superior to its commercial counterpart. Confocal imaging analysis confirmed that in the presence of 10% serum using a Lipid : DOPE of 1 : 4 and N/P charge ratio of 0.75 with 1.2 mu g DNA per well, HG-5 is better than Lipofectamine 2000.

Stimulation by DIF causes an increase of intracellular Ca2+ in Dictyostelium discoideum

Using fluorescence- activated cell sorting (FAGS), we have studied the effect of the differentiation-inducing factor (DIF) on cellular Ca2+ in Dictyostelium discoideum. We have shown previously that freshly starved or postaggregation amoebae are heterogenous with respect to the amounts of cellular Ca2+ that they contain; the L or ''low Ca2+'' class exhibits a prespore tendency and the H or ''high Ca2+'' class exhibits a prestalk tendency. Upon adding DIF, within 2 min there is an approximately twofold increase in the relative fraction of amoebae falling in the H class. A major part of the increase is caused by Ca2+ influx from the extracellular medium. Therefore a rise in the level of cellular Ca2+ is an early step in the signal transduction pathway following stimulation by DIF. Also, in parallel with the cellular heterogeneity in respect of Ca2+ content, there is a heterogeneity in the response to DIF, which appears to be restricted to L cells. (C) 1997 Academic Press.

Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+](i)) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline [0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoal, cGMP values (similar to 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/HCO(3)(-)antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 mu M) A protein kinase G inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR, A phospholipase A(2) inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+](i) was measured using fura-2-AM, there was an early rise 271 nM at 0.5 hi in pentoxifylline(-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR(20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-](i) and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A;A(2) and protein kinase C activity.

Synthesis and vesicle formation from dimeric pseudoglyceryl lipids with (CH2)(m) spacers: pronounced m-value dependence of thermal properties, vesicle fusion, and cholesterol complexation

Eight new dimeric lipids, in which the two Me2N+ ion headgroups are separated by a variable number of polymethylene units [-(CH2)(m)-], have been synthesized. The electron micrograph (TEM) and dynamic light scattering (DLS) of their aqueous dispersions confirmed the formation of vesicular-type aggregates. The vesicle sizes and morphologies were found to depend strongly on the m value, the method, and thermal history of the vesicle preparation. Information on the thermotropic properties of the resulting vesicles was obtained from microcalorimetry and temperature-dependent fluorescence anisotropy measurements. Interestingly, the T-m values for these vesicles revealed a nonlinear dependence on spacer chain length (m value). These vesicles were able to entrap riboflavin. The rates of permeation of the OH- ion under an imposed transmembrane pH gradient were also found to depend significantly on the m value. X-Ray diffraction of the cast films of the lipid dispersions elucidated the nature and the thickness of these membrane organizations, and it was revealed that these lipids organize in three different ways depending on the m value. The EPR spin-probe method with the doxylstearic acids 5NS, 12NS, and 16NS, spin-labeled at various positions of stearic acid, was used to establish, the chain-flexibility gradient and homogeneity of these bilayer assemblies. The apparent fusogenic propensities of these bipolar tetraether lipids were investigated in the presence of Na2SO4 with fluorescence-resonance energy-transfer fusion assay. Small unilamellar vesicles formed from 1 and three representative biscationic lipids were also studied with fluorescence anisotropy and H-1 NMR spectroscopic techniques in the absence and the presence of varying amounts of cholesterol.

2-Halooxyethylene ethers of cholesterol as novel single component, room temperature cholesteric LC materials

A series of 2-haloethoxyethyl cholesteryl ethers has been synthesized. Each material shows attractive liquid-crystalline properties as revealed by differential scanning calorimetry, polarizing microscopy, and temperature-dependence of selective reflection characteristic of the cholesteric mesophase. These are interesting examples of simple, nonpolymeric, single component systems that show the cholesteric mesophase at room temperature.

Advantage of the ether linkage between the positive charge and the cholesteryl skeleton in cholesterol-based amphiphiles as vectors for gene delivery

Twelve novel cationic cholesterol derivatives with different linkage types between the cationic headgroup and the cholesteryl backbone have been developed. These have been tested for their efficacies as gene transfer agents as mixtures with dioleoyl phosphatidylethanolamine (DOPE). A pronounced improvement in transfection efficiency was observed when the cationic center was linked to the steroid backbone using an ether type bond. Among these, cholest-5-en-3b-oxyethane-N, N,N-trimethylammonium bromide (2a) and cholest-5-en-3b-oxyethane-N, N-dimethyl-N-2-hydroxyethylammonium bromide (3d) showed transfection efficiencies considerably greater than commercially available reagents such as Lipofectin or Lipofectamine. To achieve transfection, 3d did not require DOPE. Increasing hydration at the headgroup level for both ester- and ether-linked amphiphiles resulted in progressive loss of transfection efficiency. Transfection efficiency was also greatly reduced when a 'disorder'-inducing chain like an oleyl (cis-9-octadecenyl) segment was added to these cholesteryl amphiphiles. Importantly, the transfection ability of 2a with DOPE in the presence of serum was significantly greater than for a commercially available reagent, Lipofectamine. This suggests that these novel cholesterol-based amphiphiles might prove promising in applications involving liposome-mediated gene transfection. This investigation demonstrates the importance of structural features at the molecular level for the design of cholesterol-based gene delivery reagents that would aid the development of newer, more efficient formulations based on this class of molecules.

Crystal and molecular structure of synthetic cholesterol compounds vitamin D3-analogues (I) 24(S)-hydroxy coprastan-3-one & (II) 24(R)-hydroxy coprastan-3-one

The title compound I (24-(S)-Hydroxy Coprastan-3-one) crystallises in orthorhombic space group P2(1)2(1)2(1) with Z = 4. The unit cell dimensions are a = 6.701(2)Angstrom, b = 11.506(8)Angstrom, c = 32.183(4)Angstrom, V = 2481(2)Angstrom (3), D-cal = 1.077 Mg/m(3). The tide compound II (24-(R)-Hydroxy Coprastan-3-one) crystallises in orthorhombic space group P212121 with two molecules per assymetric unit and with Z = 8. The Unit cell dimensions are a = 10.954(2)Angstrom, b = 21.757(6)Angstrom, c = 21.130(7)Angstrom, V = 5035.0(2)Angstrom (3), D-cal = 1.062 Mg/m(3). In compound I and in both the molecules of compound II, the rings A, B & C are in chair conformation and the five membered ring D is in envelope conformation. The priority sequence attached to the chiral carbon C24 has "S" designation in compound I and "R" designation in compound II. The structures are stabilized by C-H . . .O and O-H---O hydrogen bonds.

Synthesis and characterization of novel cationic lipid and cholesterol-coated gold nanoparticles and their interactions with dipalmitoylphosphatidylcholine membranes

Novel gold nanoparticles bearing cationic single-chain, double-chain, and cholesterol based amphiphilic units have been synthesized. These nanoparticles represent size-stable entities in which various cationic lipids have been immobilized through their thiol group onto the gold nanoparticle core. The resulting colloids have been characterized by UV-vis, (1)H NMR, FT-IR spectroscopy, and transmission electron microscopy. The average size of the resultant nanoparticles could be controlled by the relative bulkiness of the capping agent. Thus, the average diameters of the nanoparticles formed from the cationic single-chain, double-chain, and cholesterol based thiolate-coated materials were 5.9,2.9, and 2.04 nm, respectively. We also examined the interaction of these cationic gold nanoparticles with vesicular membranes generated from dipalmitoylphosphatidylcholine (DPPC) lipid suspensions. Nanoparticle doped DPPC vesicular suspensions displayed a characteristic surface plasmon band in their UV-vis spectra. Inclusion of nanoparticles in vesicular suspensions led to increases in the aggregate diameters, as evidenced from dynamic light scattering. Differential scanning calorimetric examination indicated that incorporation of single-chain, double-chain, and cholesteryl-linked cationic nanoparticles exert variable effects on the DPPC melting transitions. While increased doping of single-chain nanoparticles in DPPC resulted in the phases that melt at higher temperatures, inclusion of an incremental amount of double-chain nanoparticles caused the lowering of the melting temperature of DPPC. On the other hand, the cationic cholesteryl nanoparticle interacted with DPPC in membranes in a manner somewhat analogous to that of cholesterol itself and caused broadening of the DPPC melting transition.