Phospholipid diversity: correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called "fusion proteins". This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

Fusogenic potential of sperm membrane lipids: nature's wisdom to accomplish targeted gene delivery

The membrane-membrane fusion during fertilization of oocyte by spermatozoa is believed to be mainly mediated by so called "fusion proteins". In the present study we have tried to demonstrate that beside the proteins, lipid components of membrane may play an important role in fusion of oocyte with spermatozoa. Conventional membrane-membrane fusion assays were used as means to demonstrate fusogenic potential of human sperm membrane lipids. The liposomes (spermatosomes) made of the lipids isolated from sperm membrane were found to undergo strong membrane-membrane fusion as evident from fluorescence dequenching and resonance energy transfer assays. Furthermore, the fusion of these liposomes with living cells (J774 A.1 macrophage cell line) was demonstrated to result in an effective transfer of a water-soluble fluorescent probe (calcein) to cytosol of the target cell. Lastly, the liposomes were demonstrated to behave like efficient vehicles for the in vivo cytosolic delivery of the antigens to target cells resulting in elicitation of antigen specific CD8(+) T cell responses.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Leptin improves membrane fluidity of erythrocytes in humans via a nitric oxide-dependent mechanism--an electron paramagnetic resonance investigation

Abnormalities in physical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases. Recently, there has been an indication that leptin, the product of the human obesity gene, actively participates not only in the metabolic regulations but also in the control of cardiovascular functions. In the present study, to assess the role of leptin in the regulation of membrane properties, the effects of leptin on membrane fluidity of erythrocytes in humans are examined. The membrane fluidity of erythrocytes in healthy volunteers by means of an electron paramagnetic resonance (EPR) and spin-labeling method is determined. In an in vitro study, leptin decreased the order parameter (S) for 5-nitroxide stearate (5-NS) and the peak height ratio (ho/h-1) for 16-NS obtained from EPR spectra of erythrocyte membranes in a dose-dependent manner in healthy volunteers. The finding indicated that leptin increased the membrane fluidity and improved the microviscosity of erythrocytes. The effect of leptin on the membrane fluidity was significantly potentiated by the nitric oxide (NO) donors, L-arginine and S-nitroso-N-acetylpenicillamine (SNAP), and a cyclic guanosine monophosphate (cGMP) analog, 8-bromo-cGMP. In contrast, the change evoked by leptin was significantly attenuated in the presence of the NO synthase inhibitors, N(G)-nitro-L-arginine-methyl-ester (L-NAME) and asymmetric dimethyl-L-arginine (ADMA). The results of the present study showed that leptin increased the membrane fluidity and improved the rigidity of cell membranes to some extent via an NO- and cGMP-dependent mechanism. Furthermore, the data also suggest that leptin might have a crucial role in the regulation of rheological behavior of erythrocytes and microcirculation in humans.     

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

Structured summary of protein interactions

dicPAH and dicPAHbind by molecular sieving (View Interaction: 1, 2, 3, 4)     

Plasma membrane of Synechocystis PCC 6803: a heterogeneous distribution of membrane proteins

Proteomic studies carried out previously on the plasma membrane of Synechocystis have identified several peripheral and integral proteins. The distribution of these proteins along the membrane still remains obscure. In this study, the distribution of proteins along the plasma membrane of Synechocystis was carried out using subfractions, the right-side-out (RSO) and inside-out (ISO) vesicles, fractionated from a pure and specific fraction of the plasma membrane. These subfractions were analyzed and quantified for several proteins by immunoblotting. It was found that the ISO fraction contained higher quantities of preD1, D1 and PsaD, the integral proteins of photosystem I and II known to be present also in the plasma membrane. Lower amounts of peripheral vesicle inducing protein Vipp1 and nitrate/nitrite binding protein NrtA were present in the ISO compared to the RSO fraction. On the contrary, the distribution of two integral transporter proteins, SbtA and PxcA, was found equal in both fractions. Our studies clearly establish that the plasma membrane of Synechocystis has a heterogeneous composition with respect to protein distribution. The accumulation of photosynthesis-associated proteins in the ISO fraction provides evidence that the discrete regions of the plasma membrane harbor sites for biogenesis of photosystems.     

Phosphatidylglycerol lipids enhance folding of an alpha helical membrane protein

Membrane lipids are increasingly being recognised as active participants in biological events. The precise roles that individual lipids or global properties of the lipid bilayer play in the folding of membrane proteins remain to be elucidated, Here, we find a significant effect of phosphatidylglycerol (PG) on the folding of a trimeric α helical membrane protein from Escherichia coli diacylglycerol kinase. Both the rate and the yield of folding are increased by increasing the amount of PG in lipid vesicles. Moreover, there is a direct correlation between the increase in yield and the increase in rate; thus, folding becomes more efficient in terms of speed and productivity. This effect of PG seems to be a specific requirement for this lipid, rather than a charge effect. We also find an effect of single-chain lyso lipids in decreasing the rate and yield of folding. We compare this to our previous work in which lyso lipids increased the rate and yield of another membrane protein, bacteriorhodopsin. The contrasting effect of lyso lipids on the two proteins can be explained by the different folding reaction mechanisms and key folding steps involved. Our findings provide information on the lipid determinants of membrane protein folding.     

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.     

Effect of some nickel compounds on red blood cell characteristics

The effect of certain inorganic and coordinated nickel compounds on the resistance to different destructive substances, rheological properties, and functional activity of healthy human red blood cells (RBC), was investigated. It is shown that nickel compounds affect the erythrocyte membrane lipid bilayer, as well as membrane proteins to various extents, depending on the type of compounds used. In general, the acceleration of erythrocyte aging was observed to be more pronounced in young erythrocytes. The observed results suggest that nickel compounds decrease water permeability across erythrocyte membranes. Almost all the investigated nickel compounds decrease erythrocyte thermostability, deformability, and the rate of O₂ release by erythrocytes.     

Biophysical characterization and membrane interaction of the most membranotropic region of the HIV-1 gp41 endodomain

The membrane fusion protein of HIV-1 is the envelope transmembrane gp41 glycoprotein, which is the responsible of the membrane fusion between the virus and the target cell. Gp41 has an unusual cytoplasmic tail, the endodomain, containing highly helicoidal segments with large hydrophobic moments, the so called lentivirus lytic peptides or LLPs. According to our previous work, one of the most membranotropic regions along the whole gp41 glycoprotein was located in the LLP3 region of the gp41. In order to get new insights into the viral membrane fusion mechanism, a peptide pertaining to the LLP3 domain has been studied by infrared, fluorescence and calorimetry regarding its structure, its ability to induce membrane rupture and aggregation, as well as its affinity towards specific phospholipids. Our results demonstrate that this peptide interacts with phospholipid-containing model membranes, affects the phase-behavior of membrane phospholipids and induces leakage and aggregation of liposomes. The membrane-perturbing properties of LLP3, together with the possibility that the Kennedy sequence could be part of an external loop, open the possibility that these domains might function in modulating viral membrane fusion or budding, synergistically with other membranotropic regions of the gp41 glycoprotein.     

NMR studies of molybdate complexes of D-erythro-L-manno-octose and D-erythro-L-gluco-octose and their alditols

Different amounts and various types of bis-dinuclear tetradentate molybdate complexes of D-erythro-L-manno-octose, D-erythro-L-gluco-octose, D-erythro-L-manno-octitol and D-erythro-L-gluco-octitol were characterized by 1H and 13C NMR spectroscopy in aqueous solutions. Detailed analysis of 1H-(1)H coupling constants and NOEs, together with chemical shifts, allowed characterization of the different isomers of these complexes.     

Determination of plasma and serum l-lysine using l-lysine ε-oxidase from Marinomonas mediterranea NBRC 103028

This article describes a successful application of l-lysine ε-oxidase (EC 1.4.3.20) for l-lysine determination. l-Lysine ε-oxidase was isolated from culture supernatant of Marinomonas mediterranea NBRC 103028^T and was used for l-lysine determination. Comparison of the characteristics of l-lysine ε-oxidase with l-lysine α-oxidase, a commercial enzyme used for l-lysine determination, suggests that the use of l-lysine ε-oxidase would be more valuable for the determination of l-lysine because of its selectivity and sensitivity, especially in samples with low l-lysine concentration. The enzyme acted only on l-lysine and l-ornithine, to which the relative activity was only 3.4% of that on l-lysine. The value obtained by the colorimetric assay using l-lysine ε-oxidase and horseradish peroxidase was not affected by l-ornithine. The enzyme also shows a higher affinity for l-lysine (K_m = 0.0018 mM). l-Lysine determination using l-lysine ε-oxidase in human plasma and serum was examined. The measured values were close to values determined by instrumental analyses using the precolumn AccQ·Tag Ultra Derivatization Kit. These results suggest that l-lysine ε-oxidase can be used for diagnosis based on plasma l-lysine concentration. This is the first report on the application of l-lysine ε-oxidase.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules

We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (p<0.05). The APA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.     

Protein L-isoaspartyl methyltransferase repairs abnormal aspartyl residues accumulated in vivo in type-I collagen and restores cell migration

Abnormal aspartyl residue formation such as L-isoaspartates occurs frequently during aging in long-lived proteins, resulting in the alteration of their structures and biological functions. In this study, we investigated the alteration of aspartyl residues in extracellular matrix (ECM) proteins, type-I collagen and fibronectin, and in integrin- and ECM-binding motifs during aging, as well as the resulting effects on cell biological functions such as migration and attachment. Using protein L-isoaspartyl methyltransferase (PIMT) to monitor the presence of L-isoaspartyl residues, we showed their accumulation during in vivo aging in type-I collagen from rats. In vitro aging of fibronectin as well as of peptides containing an integrin- or ECM-binding motif such as RGDSR, KDGEA and KDDL also resulted in the formation of L-isoaspartyl residues. While aged fibronectin does not alter cell adhesion and migration, type-I collagen aged 20 months reduced by 65% cell motility, but not adhesion, when compared to 3-month-aged type-I collagen. Finally, by repairing 20-month-old type-I collagen with recombinant PIMT (rPIMT), cell migration was recovered by 72%. These results strongly suggest that L-isoaspartyl residue formation in ECM proteins such as type-I collagen could play an important role in reducing cell migration and that PIMT could be a therapeutic tool to restore normal cell migration in pathological conditions where cell motility is crucial.     

Efficient synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) from L-arabinose

An efficient and practical route for the large-scale synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) starting from L-arabinose was developed using Barton-type free-radical deoxygenation reaction as a key step. The radical precursor, a phenoxythiocarbonyl ester, was prepared in situ, and the most efficient deoxygenation was achieved by slow addition of tributyltin hydride to the reaction mixture.     

Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate beta-lyase

Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.