Palmitoyl Research Articles

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A(2) treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.

The encapsulation of bleomycin within chitosan based polymeric vesicles does not alter its biodistribution.

Polymeric vesicles have recently been developed from an amphiphilic chitosan derivative--palmitoyl glycol chitosan. Their potential as a drug delivery system was evaluated using the anti-cancer compound bleomycin as a model drug. Palmitoyl glycol chitosan (GCP41) was synthesised by conjugation of palmitoyl groups to glycol chitosan. Bleomycin-containing vesicles (669 nm diameter) were prepared from a mixture of GCP41 and cholesterol by remote loading. The vesicles were imaged by freeze-fracture electron microscopy and their in-vitro stability tested. Incubation of the larger vesicles with plasma in-vitro led to a reduction of mean size by 49%, a reaction not seen with control sorbitan monostearate niosomes (215 nm in size). They also showed a higher initial drug release (1 h), but GCP41 and sorbitan monostearate vesicles retained 62% and 63% of the encapsulated drug after 24h, respectively. The biodistribution of smaller vesicles (290 nm) prepared by extrusion through a 200-nm filter was also studied in male Balb/c mice. Encapsulation of bleomycin into polymeric vesicles did not significantly alter the pharmacokinetics of biodistribution of bleomycin in male Balb/c mice although plasma and kidney levels were slightly increased. It is concluded that the extruded GCP41 vesicles break down in plasma in-vivo and hence are unlikely to offer any therapeutic advantage over the free drug.

Highlights From The Literature

Highlights From The Literature

Surface Rheological and Morphological Studies of Peptoid Mimics of Lung Surfactant Protein C

Surfactant protein C (SP-C) is a lipoprotein secreted by alveolar type II cells that has been implicated in surface-associated activities thought to facilitate breathing and to prevent alveolar collapse. The N-terminal cysteine residues of SP-C are palmitoylated, which is thought to be critical in stabilizing the helical structure and maintaining a surface-associated surfactant reservoir. However, the exact function of the two palmitoyl chains is not yet fully understood. In the current study, poly-N-substituted glycines or “peptoids”, a class of novel bio-inspired foldamers, have been employed to study the effects of N-terminal alkylation of a petpoid-based mimic of SP-C. Langmuir isotherms were performed to examine the reversibility of non-alkylated and di-alkylated SP-C mimic-containing lipid films during compression and expansion cycles at the air/liquid interface. Atomic force microscopy (AFM) of Langmuir-Blodgett films revealed extensive multilayer formation at high compression for a lipid film of dipalmitoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylglycerol and palmitic acid with the presence of di-alkylated SP-C mimics, in contrast to the film containing the non-alkylated SP-C mimics. Interfacial stress rheometor (ISR) measurements show that the lipid system with non-alkylated SP-C mimics displays a sharp increase in loss modulus at a surface pressure of ∼42mN/m, corresponding to the plateau region in the isotherms. The dramatic increase in surface viscosity is consistent with the increase in the fraction of solid phase in the AFM images. In contrast, di-alkylated SP-C-containing films are more fluid at high surface pressures with a moderate increase in viscosity, suggesting an important role of the di-alkylated chains to associate with lipid acyl chains and maintain the coexistence of both fluid-like and solid phases at high surface pressures.

Solid State NMR Studies on Acylated Transmembrane Peptides Driving Membrane Fusion

The fusion of biological membranes is mediated by integral membrane proteins with α-helical transmembrane segments (TMSs). Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed α-helical peptides with mixed Leu/Val sequences was presented, mimicking fusogenic TMSs in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776-14781). From this series, we have investigated the peptide LV16 (KKKWLVLVLVLVLVLVLVLVKKK), which was synthesized presenting either a free N-terminus or an N-acylation of 2, 8, 12, or 16 carbons. We used 2H and 31P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C-2 chain, all peptide acyl chains were found to insert well into the membrane. This can be understood from the high local lipid concentration, which the N-terminal lipid chains experience. The insertion of these peptides did not influence the membrane structure and dynamics as seen from 2H and 31P NMR. In spite of the fact that the longer acyl chains insert into the membrane, there is no perfect length adaptation. Even the C-16 chain on the peptide, which could match the length of the POPC palmitoyl chain exhibited lower order parameters in the upper chain, which get closer and finally reach similar values in the lower chain region. 2H NMR square law plots reveal a slightly more dynamic characteristic of the peptide acyl chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acyl chains, the fraction of gauche defects in the inserted chains is constant, suggesting similar entropies of the inserted chains.

Investigating the Nanodomain Structure of Sphingomyelin/Cholesterol Membranes Using 2H NMR

We have used 2H NMR to study the dependence of the phase and nanodomain structure of sphingomyelin/cholesterol membranes on sterol content and temperature. NMR spectra of N-palmitoyl (D31)-D-erythro-sphingosylphosphorylcholine (PSM) were taken for temperatures from 25 to 70°C and cholesterol concentrations 0 - 40%. Three distinct phases are observed: solid-ordered (so), liquid-disordered (ld) and liquid-ordered (lo). The constructed phase diagram exhibits both so+lo and ld +lo phase coexistence regions, however macroscopic (micron-sized) coexistence of ld + lo does not occur. Instead, we observed line-broadening in the ld +lo region which was characterized by examining the cholesterol dependence of the quadrupolar splittings and linewidths of the peaks in the depaked spectra, at a given temperature. These observations were analyzed in terms of fast exchange of lipids between ld and lo nanodomains. However, by analyzing the depaked spectrum, information from all orientations of the lipid long axis, which constitutes the powder pattern, is pooled. This may obscure the results used to characterize domain size. To attempt to gain new insight into the structural properties of the domains in PSM/chol membranes we used selectively deuterated sphingomyelin, labeled at C9 of the N-linked palmitoyl chain, to measure the orientation dependence of T2 relaxation. NMR measurement of T2 relaxation time shows that it does depend on the orientation of the axis of symmetry of the motion - the lipid long axis - relative to the magnetic field. For example, for small angles T2 is found to be as short as 200μs whereas the 90 degree orientation has a relaxation time longer than 600μs. We will present data on the orientation-specific influence of cholesterol on the sphingomyelin spectrum's relaxation time and discuss lo + ld nanodomain structure in this context.

The Essential Escherichia coli Apolipoprotein N-Acyltransferase (Lnt) Exists as an Extracytoplasmic Thioester Acyl-Enzyme Intermediate

Escherichia coli apolipoprotein N-acyltransferase (Lnt) transfers an acyl group from sn-1-glycerophospholipid to the free alpha-amino group of the N-terminal cysteine of apolipoproteins, resulting in mature triacylated lipoprotein. Here we report that the Lnt reaction proceeds through an acyl-enzyme intermediate in which a palmitoyl group forms a thioester bond with the thiol of the active site residue C387 that was cleaved by neutral hydroxylamine. Lnt(C387S) also formed a fatty acyl intermediate that was resistant to neutral hydroxylamine treatment, consistent with formation of an oxygen-ester linkage. Lnt(C387A) did not form an acyl-enzyme intermediate and, like Lnt(C387S), did not have any detectable Lnt activity, indicating that acylation cannot occur at other positions in the catalytic domain. The existence of this thioacyl-enzyme intermediate allowed us to determine whether essential residues in the catalytic domain of Lnt affect the first step of the reaction, the formation of the acyl-enzyme intermediate, or the second step in which the acyl chain is transferred to the apolipoprotein substrate. In the catalytic triad, E267 is required for the formation of the acyl-enzyme intermediate, indicating its role in enhancing the nucleophilicity of C387. E343 is also involved in the first step but is not in close proximity to the active site. W237, Y388, and E389 play a role in the second step of the reaction since acyl-Lnt is formed but N-acylation does not occur. The data presented allow discrimination between the functions of essential Lnt residues in catalytic activity and substrate recognition.

The Molecular Shape of Poly(propylenimine) Dendrimer Amphiphiles Has a Profound Effect on Their Self Assembly

The shape of dendrimer amphiphiles has an unexpected effect on their self-assembly. A series of diaminobutane poly(propylenimine) generation 3 dendrimer (DAB-dendr-(NH(2))(16)) amphiphiles has been synthesized, bearing an average of five (PD5), three (PD3) and one (PD1) palmitoyl group(s) per dendrimer molecule. Additionally DAB-dendr-(NH(2))(16) was derivatized with a layer of poly(ethylene glycol) (PEG, degree of polymerization = 12) groups and conjugated to an average of 1 palmitoyl group at the PEG end (PPD1). A final amphiphile resulted from the conjugation of DAB-dendr-(NH(2))(16) with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-succinimidylpropionate (DSPE-PEG(3400)-SPA), i.e.: DPD5 (with 4 DSPE-PEG arms). The critical micellar concentration in aqueous media followed the trend: DPD5 < PD5 = PD3 < PD1 < PPD1 and amphiphiles eventually formed 10-20 nm monomolecular or multimolecular micelles and/or 200 nm spheres or tubules. Aggregation was entropy driven, as expected, for DPD5, PD5 and PD1 and enthalpy driven with the most hydrophilic compound PPD1, but was unexpectedly enthalpy driven for PD3. PD3 aggregates formed low capacity hydrophobic domains with a limited capacity for encapsulation of cyclosporine A; encapsulation levels (mole drug per mole polymer) were 0.099, 0.014, 0.099, and 0.735 for PD1, PD3, PD5, and DPD5 and, respectively. We conclude that star shaped amphiphiles such as PD3 are sterically hindered from self-assembling into high capacity hydrophobic domains in aqueous media. Amphiphile-membrane interactions were promoted by hydrophobic groups, but diminished by PEG moieties. DPD5 is the most suitable amphiphile for biomedical applications.

Orientation of palmitoylated CaVβ2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation

The Gq-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca2+ channel (CaV2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which CaVβ is coexpressed with CaV2.2. When palmitoylated CaVβ2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when CaVβ3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of CaVβ2a may occupy an inhibitory site on CaV2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of CaVβ2a relative to CaV2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of CaVβ2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the α interacting domain (AID) of CaV2.2's I–II linker. CaVβs bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple CaVβ's movements to CaV2.2 gating. Although Bdel1/β2a currents exhibited more variable enhancement by SP, Bdel2/β2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from CaV2.2 coexpressed with CaVβ3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/β2a currents, suggesting that when palmitoylated CaVβ2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that CaVβ2a's palmitoyl groups directly interact with an inhibitory site on CaV2.2 to block N-current inhibition by SP.

A new force field for simulating phosphatidylcholine bilayers

A new force field for the simulation of dipalmitoylphosphatidylcholine (DPPC) in the liquid-crystalline, fluid phase at zero surface tension is presented. The structure of the bilayer with the area per lipid (0.629 nm(2); experiment 0.629-0.64 nm(2)), the volume per lipid (1.226 nm(3); experiment 1.229-1.232 nm(3)), and the ordering of the palmitoyl chains (order parameters) are all in very good agreement with experiment. Experimental electron density profiles are well reproduced in particular with regard to the penetration of water into the bilayer. The force field was further validated by simulating the spontaneous assembly of DPPC into a bilayer in water. Notably, the timescale on which membrane sealing was observed using this model appears closer to the timescales for membrane resealing suggested by electroporation experiments than previous simulations using existing models.

Molecular Mechanism for Lateral Lipid Diffusion between the Outer Membrane External Leaflet and a β-Barrel Hydrocarbon Ruler

Membrane-intrinsic enzymes are embedded in lipids, yet how such enzymes interrogate lipid substrates remains a largely unexplored fundamental question. The outer membrane phospholipid:lipid A palmitoyltransferase PagP combats host immune defenses during infection and selects a palmitate chain using its beta-barrel interior hydrocarbon ruler. Both a molecular embrasure and crenel in Escherichia coli PagP display weakened transmembrane beta-strand hydrogen bonding to provide potential lateral routes for diffusion of the palmitoyl group between the hydrocarbon ruler and outer membrane external leaflet. Prolines in strands A and B lie beneath the dynamic L1 surface loop flanking the embrasure, whereas the crenel is flanked by prolines in strands F and G. Reversibly barricading the embrasure prevents lipid A palmitoylation without affecting the slower phospholipase reaction. Lys42Ala PagP is also a dedicated phospholipase, implicating this disordered L1 loop residue in lipid A recognition. The embrasure barricade additionally prevents palmitoylation of nonspecific fatty alcohols, but not miscible alcohols. Irreversibly barricading the crenel inhibits both lipid A palmitoylation and phospholipase reactions without compromising PagP structure. These findings indicate lateral palmitoyl group diffusion within the PagP hydrocarbon ruler is likely gated during phospholipid entry via the crenel and during lipid A egress via the embrasure.

PapA3 Is an Acyltransferase Required for Polyacyltrehalose Biosynthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.

Effect of NaCl and CaCl 2 on the lateral diffusion of zwitterionic and anionic lipids in bilayers

The influence of addition of NaCl or CaCl 2 (0.3 and 0.1 M, respectively) on the lateral diffusion coefficient ( D L ) of dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) was measured by the pulsed field gradient NMR technique. D L of DOPC was unaffected, whereas the DOPG diffusion decreased with salt concentration. 23Na NMR quadrupole splittings of DOPG between 20 and 60 °C and added NaCl between 0 and 15 wt% decreased only slightly with salt content, but increased with increasing temperature. Similar results were obtained for palmitoyloleoylphosphatidylglycerol, in which the palmitoyl chain order parameter increased slightly with salt. A model with free and “bound” ions was used to interpret the splitting data. With increasing salt content a decrease in the water permeability for DOPG was observed, but not for DOPC, as measured by water diffusion perpendicular to the oriented lipid bilayers. It was concluded that calcium and sodium ions interacted with the DOPG head-groups resulting in a decrease in the “free area” per lipid molecule due to a screening of the charged lipid head-groups. Thus, there was a closer packing of DOPG, leading to a decrease in D L and water permeability. DOPC did not show any changes in the bilayer properties upon the addition of ions.

Arachidonic acid inhibition of L-type calcium (CaV1.3b) channels varies with accessory CaVβ subunits

Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca2+ channels by an unknown mechanism at an unknown site. The Ca2+ channel pore-forming subunit (CaVα1) is a candidate for the site of AA inhibition because T-type Ca2+ channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory CaVβ subunits on the inhibition of CaV1.3b L-type (L-) current by AA. Whole cell Ba2+ currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of −10 mV from a holding potential of −90 mV. A one-minute exposure to 10 µM AA inhibited currents with β1b, β3, or β4 58, 51, or 44%, respectively, but with β2a only 31%. At a more depolarized holding potential of −60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes CaV1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential–dependent inactivation or on recovery from inactivation regardless of CaVβ subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for CaV2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of β2a interfere with inhibition by AA. Our novel findings that the CaVβ subunit alters kinetic changes and magnitude of inhibition by AA suggest that CaVβ expression may regulate how AA modulates Ca2+-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.

The Physical Properties of Sphingomyelin/Cholesterol Membranes: a Deuterium NMR Study to Map a Partial Phase Diagram

We have used deuterium nuclear magnetic resonance (2H NMR) to study the effect of cholesterol on N-palmitoyl(D31)-D-erythro-sphingosylphosphorylcholine (PSM) membranes. NMR spectra were taken as a function of temperature (from 25 to 70°C) and cholesterol concentration (0 - 40%). The constructed phase diagram exhibits both solid-ordered (so) + liquid-ordered (lo) and liquid-disordered (ld) + lo phase coexistence regions with a clear three-phase line at 37.5°C between 8 and approx. 18 mol% cholesterol. The ld + lo region was characterized by examining the cholesterol dependence of the width of resolved peaks in the depaked spectra, as well as the average spectral width (M1), at a given temperature. The so + lo region was defined using spectral subtraction. Analogous experiments were done using 1-palmitoyl,2-palmitoyl(D31)-sn-glycero-3-phosphocholine (DPPC)/cholesterol membranes in order to carefully compare the data obtained using palmitoyl chains which have similar “kinked” conformations. The three-phase line in sn-2 perdeuterated DPPC/cholesterol is at 39.5°C and extends from approx. 8 to 20 mol% sterol. The PSM/cholesterol membrane is significantly more ordered than the DPPC/cholesterol membrane in the liquid crystalline phase. For example, at 60°C and 20 mol% cholesterol, the average order parameter of PSM is 34% higher than for DPPC. This should be compared to the difference between the cholesterol-free membranes at 60°C, where the average order of PSM is 23% more than that of DPPC. However the PSM/chol interaction is more effective than that of DPPC/chol at removing conformational fluctuations in the palmitoyl chain.

One membrane protein, two structures and six environments: a comparative molecular dynamics simulation study of the bacterial outer membrane protein PagP

PagP is a bacterial outer membrane protein consisting of an 8 stranded transmembrane beta-barrel and an N-terminal alpha-helix. It is an enzyme which catalyses transfer of a palmitoyl chain from a phospholipid to lipid A. Molecular dynamics simulations have been used to compare the dynamic behaviour in simulations starting from two different structures (X-ray vs. NMR) and in six different environments (detergent micelles formed by dodecyl phosphocholine and by octyl glucoside, vs. four species of phospholipid bilayer). Analysis of interactions between the protein and its environment reveals the role played by the N-terminal alpha-helix, which interacts with the lipid headgroups to lock the PagP molecule into the bilayer. The PagP beta-barrel adopts a tilted orientation in lipid bilayers, facilitating access of lipid tails into the mouth of the central binding pocket. In simulations starting from the X-ray structure in lipid bilayer, the L1 and L2 loops move towards one another, leading to the formation of a putative active site by residues H33, D76 and S77 coming closer together.

Apolipoprotein E peptide-modified colloidal carriers: The design determines the mechanism of uptake in vascular endothelial cells

Supramolecular structures, particularly micelles and liposomes equipped with uptake-mediating address compounds, have attracted much attention as pharmaceutical formulations. Their development requires an understanding of the mechanism by which the carrier systems interact with and translocate into the target cells. We developed an apolipoprotein E-derived peptide, called A2, that efficiently translocates across cell membranes. Upon coupling of two palmitoyl chains (P2), the highly cationic sequence acquires detergent-like properties such as a strong tendency to self-associate and the ability to integrate into lipid bilayers. Confocal laser scanning microscopy and fluorescence activated cell sorting were used to compare the internalization of the fluorescence-labeled monomeric A2 with the uptake of the colloidal P2A2 micelles and P2A2-tagged liposomes into endothelial cells of blood vessels. Specific inhibitors of endocytosis were used to identify the underlying mechanisms. b.End3 and BAEC cells as example of endothelial cells of small capillaries and large vascular vessels, respectively, were examined. The uptake of monomeric A2 was characterized by poor cellular selectivity. A2 was efficiently internalized into both cell lines via at least two different mechanisms. Besides an endocytotic uptake route, a second passive pathway exists, that leads to a rapid distribution of A2 within the cytoplasm. Also liposomes tagged with P2A2 were non-selectively internalized into both b.End3 and BAEC cells. Their nonselective uptake was mediated by clathrin- and caveolin-independent endocytosis. In contrast, micellar P2A2 entered b.End3 cells via clathrin-mediated endocytosis, while no uptake of P2A2 into BAEC cells was observed. In conclusion, the specific clathrin-mediated uptake mode of P2A2 micelles might provide the basis for a blood brain barrier-specific targeting.

Iron (III) porphyrin bearing 2,6-di- tert-butylphenol pendants deposited onto gold electrodes for amperometric determination of l-histidine

A sensitive amperometric sensor for determination of l-histidine was developed using gold electrode modified with Fe(III)-porphyrin bearing three 2,6-di- tert-butylphenol groups and one palmitoyl chain. Two methods of electrode modification were applied: direct chemisorption and embedment into dodecanethiol monolayer. Both types of electrodes were used for detection of l-histidine using Osteryoung square-wave voltammetry. The sensitivity of sensors presented towards l-histidine depends on the method of electrode modification. The detection limits observed for the electrodes incorporating with Fe(III)-porphyrin host by embedment and chemisorption were in 1 and 100 nM ranges, respectively. In addition, the determination of l-histidine with electrode modified by embedment technique was more precise, in comparison to that obtained by the direct chemisorption. Applicability of gold electrodes modified with Fe(III)-porphyrin for the direct electrochemical determination of l-histidine was demonstrated using the artificial matrix mimicking human serum.

Enhanced antitumor activities of (−)-epigallocatechin-3-O-gallate fatty acid monoester derivatives in vitro and in vivo

(−)-Epigallocatechin-3-O-gallate (EGCG) monoesters modified with butanoyl (EGCG-C4), octanoyl (EGCG-C8), palmitoyl groups (EGCG-C16) were synthesized by a lipase-catalyzed transesterification method and their antitumor activities were investigated in vitro and in vivo. The in vitro antitumor activities of EGCG-monoester derivatives increased in an alkyl chain length-dependent manner. The cytotoxicity of EGCG, EGCG-C4, EGCG-C8 was mainly caused by H2O2 which was generated with their oxidation. On the other hand, EGCG-C16 was more stable than EGCG and it did not generate H2O2 in the cell culture medium. Furthermore, EGCG-C16 inhibited cell proliferation and induced apoptosis in the presence of catalase. EGCG-C16 was found to inhibit the phosphorylation of the epidermal growth factor receptor (EGFR), which is related to various types of tumor growth. EGCG-C16 suppressed tumor growth in vivo in colorectal tumor bearing mice in comparison to an untreated control, vector control (DMSO) and EGCG.

A Novel Interaction Linking the FAS-II and Phthiocerol Dimycocerosate (PDIM) Biosynthetic Pathways

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.