Electrospun phospholipid polymer substrate for enhanced performance in immunoassay system

A functional polymer bearing both phosphorylcholine and active ester groups, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-N-succinimidyloxycarbonyl di(ethylene glycol) methacrylate (PENHS)] (PMBS), provided a highly biomolecule-friendly platform for immunoassays. The nonbiofouling property of the PMBS remarkably reduces the background noise from nonspecific adsorption of proteins in the analyte in an enzyme-linked immunosorbent assay (ELISA), which improves the specificity and signal-to-noise ratio. Electrospinning deposition, a versatile and cost-effective technique, was employed to fabricate nanofibrous PMBS. This nanostructure increased the surface area of the polymer, allowing more antibodies to bind to the polymer interface and enhancing the sensitivity of the biosensing system. The electrospun PMBS fibers were stable and retained their unique morphologies after contact with an aqueous solution for 4.0h. The ability of PMBS to reduce background noise without blocking by protein-based reagents was verified by comparison with an immunoassay conducted on a polystyrene substrate. The ELISA of human immunoglobulin-G with the electrospun PMBS substrate showed a good sigmoidal relationship with a linear detection range from 1.0 to 100ng/mL. The detection time was 25% shorter than the conventional assay as the blocking step was omitted. The immobilized primary antibodies exhibited high stability on the electrospun PMBS; 60% and 25% of the residual bioactivity remained after storage in dry conditions for 2 and 4 weeks, respectively. Thus, for further development of biosensors, nanostructured PMBS can improve lifetime of immobilized biomolecules, and also contribute to an enhanced reliability and signal-to-noise ratio of immunoassay.     

Biosynthesis of paf-acether. XI. Regulation of acetyltransferase by enzyme-substrate imbalance

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase is the key enzyme in paf-acether (paf) biosynthesis, since it yields the active mediator from its nonacetylated precursor, lyso-paf. In microsomal fractions obtained from the ionophore A23187-stimulated human polymorphonuclear neutrophils, the optimal conditions allowing the full acetylation of lyso-paf were: 2-2.5 mg.ml-1 bovine serum albumin, 40 microM lyso-paf, 200 microM acetyl-CoA and acetyltransferase of high specific activity, at least 18 nmol.min-1.mg protein- -1. The reaction frequently stopped before the substrate was consumed due to spontaneous decay of the enzyme activity at 37 degrees C and inhibition of the enzyme by the paf formed in the reaction. However, low concentrations of acetyltransferase substrates (lyso-paf or lysophosphatidylcholine) and the antioxidant dithiothreitol, but not the inhibitors of proteinases or phosphatases, protected the enzyme against decay. In contrast, high concentrations of those lyso substrates inhibited the enzyme activity in the assay. This inhibition as well as that due to paf was overcome by raising the concentration of the enzyme contained in the microsomal fraction or the bovine serum albumin in the assay. These results suggest that the biosynthesis of paf in cell-free assay and most probably in intact cells might be controlled to a larger extent by the acetyltransferase concentration rather than by that of its substrates.     

Regulation of mitogenesis by water-soluble phospholipid intermediates.

Many recent observations implicate choline and ethanolamine kinases as well as phosphatidylcholine-specific phospholipase C in the regulation of mitogenesis and carcinogenesis. For example, human cancers generally contain high concentrations of phosphoethanolamine and phosphocholine, and in different cell lines various growth factors, cytokines, oncogenes and chemical carcinogens were all shown to stimulate the formation of phosphocholine and phosphoethanolamine. In addition, other reports have appeared showing that both extracellular and intracellular phosphocholine as well as ethanolamine and its derivatives can regulate cell growth. This area of research has clearly arrived at a stage when it becomes important to examine critically the feasibility of water-soluble phospholipid intermediates serving as potential regulators of cell growth in vivo. Accordingly, the goal of this review is to summarise available information relating to the formation and mitogenic actions of intracellular and extracellular phosphocholine as well as ethanolamine and its derivatives.     

Regulation of phosphatidylethanolamine methyltransferase and phospholipid methyltransferase by phospholipid precursors in Saccharomyces cerevisiae

Phosphatidylethanolamine methyltransferase (PEMT) and phospholipid methyltransferase (PLMT), which are encoded by the CHO2 and OPI3 genes, respectively, catalyze the three-step methylation of phosphatidylethanolamine to phosphatidylcholine in Saccharomyces cerevisiae. Regulation of PEMT and PLMT as well as CHO2 mRNA and OPI3 mRNA abundance was examined in S. cerevisiae cells supplemented with phospholipid precursors. The addition of choline to inositol-containing growth medium repressed the levels of CHO2 mRNA and OPI3 mRNA abundance in wild-type cells. The major effect on the levels of the CHO2 mRNA and OPI3 mRNA occurred in response to inositol. Regulation was also examined in cho2 and opi3 mutants, which are defective in PEMT and PLMT activities, respectively. These mutants can synthesize phosphatidylcholine when they are supplemented with choline by the CDP-choline-based pathway but they are not auxotrophic for choline. CHO2 mRNA and OPI3 mRNA were regulated by inositol plus choline in opi3 and cho2 mutants, respectively. However, there was no regulation in response to inositol when the mutants were not supplemented with choline. This analysis showed that the regulation of CHO2 mRNA and OPI3 mRNA abundance by inositol required phosphatidylcholine synthesis by the CDP-choline-based pathway. The regulation of CHO2 mRNA and OPI3 mRNA abundance generally correlated with the activities of PEMT and PLMT, respectively. CDP-diacylglycerol synthase and phosphatidylserine synthase, which are regulated by inositol in wild-type cells, were examined in the cho2 and opi3 mutants. Phosphatidylcholine synthesis was not required for the regulation of CDP-diacylglycerol synthase and phosphatidylserine synthase by inositol.     

Effect of phospholipid on substrate phosphorylation by a catalytic fragment of protein kinase C

Limited tryptic digestion of protein kinase C purified from mouse brain generated a 36-kDa fragment which no longer required Ca2+ and phospholipid for activity or bound phorbol ester. Under appropriate conditions, the isolated fragment was stable for several months at 4 degrees C or upon freezing and storage at -70 degrees C. Kinetic characteristics of the fragment were similar to those for the intact protein kinase. Although the fragment did not require phospholipid for activity, anionic phospholipids affected the extent of its activity in a pH-, substrate-, and substrate concentration-dependent manner. This effect appeared to be due to complex formation between the phospholipid and substrate. The catalytic fragment thus permits detection of a second point of interaction of phospholipid with the protein kinase C system in addition to the already described phospholipid regulatory domain.     

Regulation of the Golgi complex by phospholipid remodeling enzymes

The mammalian Golgi complex is a highly dynamic organelle consisting of stacks of flattened cisternae with associated coated vesicles and membrane tubules that contribute to cargo import and export, intra-cisternal trafficking, and overall Golgi architecture. At the morphological level, all of these structures are continuously remodeled to carry out these trafficking functions. Recent advances have shown that continual phospholipid remodeling by phospholipase A (PLA) and lysophospholipid acyltransferase (LPAT) enzymes, which deacylate and reacylate Golgi phospholipids, respectively, contributes to this morphological remodeling. Here we review the identification and characterization of four cytoplasmic PLA enzymes and one integral membrane LPAT that participate in the dynamic functional organization of the Golgi complex, and how some of these enzymes are integrated to determine the relative abundance of COPI vesicle and membrane tubule formation. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Regulation of eukaryotic phospholipid metabolism

Phospholipids have diverse and critical roles in cellular metabolism and function. Questions about the mechanisms of regulation of phospholipid synthesis are being investigated with a variety of systems and approaches. For example, the yeast Saccharomyces cerevisiae is an organism in which both biochemical and genetic analyses are used. Biochemical approaches have yielded considerable information on the regulatory properties of enzymes of phospholipid biosynthesis. Studies of the activity of purified phosphatidylserine synthase have suggested how that enzyme is influenced by membrane phospholipids in the cell. The enzyme that regulates mammalian phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, is also influenced by phospholipids. In addition, the activity of this enzyme often correlates with its translocation to membranes. The location of such enzymes in the cell is of particular interest in light of the possibility that the enzymatic reactions may be efficiently coupled in vivo. Techniques to render cultured cells permeable to phosphorylated molecules indicated that the enzymes of phosphatidylcholine biosynthesis may exist in an organized compartment so that the precursors of phosphatidylcholine are efficiently channeled through the pathway. To ask how phospholipids are transported in the cell, a combined biochemical and genetic approach has been used. These studies have revealed that the phosphatidylinositol/phosphatidylcholine transfer protein, considered to mediate intracellular phospholipid transfer, is a critical component of the secretory pathway for proteins. These results have allowed formulation of a number of new questions on the regulation of phospholipid metabolism and its relationship to general membrane processes.     

Gas-filled phospholipid nanoparticles conjugated with gadolinium play a role as a potential theragnostics for MR-guided HIFU ablation

To develop a long-circulating theragnostics, meaning therapeutics and diagnostics for MR-guided HIFU ablation, we designed and prepared Gd-C₅F₁₂-phospholipid nanobubbles (PLNs) 30–100 nm in diameter. The biochemical and physical characterization of Gd-C₅F₁₂-PLNs were performed. Since Gd-C₅F₁₂-PLN-50 (Φ = 50 nm) and Gd-C₅F₁₂-PLN-100 (Φ = 100 nm) enhanced the hyperthermal effect of HIFU size- and concentration-dependently in a tissue-mimicking phantom, its circulation, distribution, tumor accumulation and tumor ablation were examined in tumor-bearing mice. The plasma-half life of Gd-C₅F₁₂-PLNs was longer than 1.5 hrs. Gd-C₅F₁₂-PLNs mainly accumulated in the liver and the spleen, suggesting that they are slowly secreted through the hepatobiliary pathway. Monitored by the T1 signal intensity of MR, Gd-C₅F₁₂-PLNs accumulated in tumor tissues for 8 hours in mice. HIFU with Gd-C₅F₁₂-PLN-100 showed the increased tumor ablation area as compared with HIFU alone. The results suggest that Gd-C₅F₁₂-PLNs exhibit a potential theragnostics for MR-guided HIFU ablation.     

The cyanogenic substrate for horseradish peroxidase is a conjugated enamine

We identify the cyanogenic substrate for horseradish peroxidase (HRP) as a conjugated enamine and explore this unusual reaction using alpha-aminocinnamate (RH) as follows. 1) HRP catalyzes the oxidation of RH by O2 (and its peroxidation by H2O2 to form R-R) to produce, simultaneously, CN- and benzaldehyde cyanohydrin. 2) RH is transient and must be generated in situ. The properties of the cyanogenic reaction of HRP are independent of the method of preparation of RH (whether this be condensation of NH3 with phenylpyruvate, enzymatic hydrolysis of glycyldehydrophenylalanine, or oxidation of L-phenylalanine by L-amino acid oxidase). 3) The oxidation of RH is a free radical chain reaction initiated by HRP Compounds I and II (I (or II) + RH----R. + II (or HRP], propagated by RO2. (R. + O2----RO2., RO2. + RH----R. + RO2H), and terminated by recombination reactions such as 2R.----R2 and RO2.----R' + HO2. followed by R. + HO2.----RH + O2. KMnO4 and K3Fe(CN)6 can substitute for HRP. 4) The proximal precursor of CN- and cyanohydrin is postulated to be RO2H (phi-CH(-O2H)-CCO2-(= NH]. These results explain why cyanide is generated from the synergistic action of HRP and L-amino acid oxidase on aromatic L-amino acids and O2 and suggest that the requirement for a beta-aryl substituent on the enamine originates in the reaction of RH with HRP, or of R with O2, rather than the imine/enamine tautomerization of the L-amino acid oxidase product.     

Regulation of growth and adhesion of cultured cells by insulin conjugated with thermoresponsive polymers

We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1-10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 339-344, 1997.     

Structural requirements of the phospholipid substrate for phospholipid N-methylation in rat liver

The role of endogenous phospholipid substrates for phospholipid methylation was investigated in rat liver microsomes. The amount of phosphatidylethanolamine could be drastically reduced by treatment of microsomes with an amino group-blocking compound, methylacetimidate. Simultaneously, the formation of labelled phospholipids from S-adenosyl[Me-3H]methionine decreased, indicating that the amount of endogenous substrate influenced the reaction rate. Phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and phosphatidylmonoethylethanolamine added as dispersions to untreated or treated microsomes stimulated phospholipid methylation, whereas several other phospholipids were inactive. In other experiments the role of phospholipid substrates in intact cells was studied. Cultured rat hepatocytes were enriched in different phospholipids by preincubation with different amino alcohols, and the effects of phospholipid methylation was measured by incubation with [Me-14C]methionine. Phospholipid methylation was significantly stimulated after preincubation with ethanolamine, monomethylethanolamine, monoethylethanolamine and 2-aminobutanol. The results show that both the number and chain length of N-alkyl substituents on phosphatidylethanolamine, as well as other changes in the ethanolamine moiety, will affect the ability of different phospholipids to act as methyl acceptors.     

Regulation of phospholipid synthesis inMycobacterium smegmatis by cyclic adenosine monophosphate

Forskolin, an adenylate cyclase activator and a cyclic AMP analogue, dibutyryl cyclic AMP have been used to examine the relationship between intracellular levels of cyclic AMP and lipid synthesis inMycobacterium smegmatis. Total phospholipid content was found to be increased in forskolin grown cells as a result of increased cyclic AMP levels caused by activation of adenylate cyclase. Increased phospholipid content was supported by increased [¹⁴C] acetate incorporation as well as increased activity of glycerol-3-phosphate acyltransferase. Pretreatment of cells with dibutyryl cyclic AMP had similar effects on lipid synthesis. Taking all these observations together it is suggested that lipid synthesis is being controlled by cyclic AMP in mycobacteria.     

Regulation of phospholipid synthesis in Escherichia coli by guanosine tetraphosphate

Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with (32)P-inorganic phosphate and (32)P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells.     

Urease inhibition by polymer analogs of substrate and thiophosphamides

Inhibition of soybean urease by polymeric substrate analogues, urea and thiourea polydisulfides (PDSU and PDSTU, respectively), or three thiophosphoric acid amides (TPAA), tri-(N-3-hydroxyphenyl)thiophosphamide (1), tri-(N-4,4'-aminodiphenyl)thiophosphamide, and di-oxy-(N-alpha-piridyl)thiophosphamide (3) was studied in aqueous solutions at various pH values. The inhibitory effects of all these substances were reversible and competitive with the lowest inhibition constant Ki 2.8 microM for TPAA-1 at pH 3.85. Above and below this pH value, Ki increased reaching 24 [mu]M at pH 7.2. All test substances inhibited urease comparably with known inhibitors such as thiols (cysteamine, etc.) and hydroxamic acid derivatives, but were less efficient than phosphorodiamidates. Structural features of possible urease inhibitors of higher efficiency were proposed.     

Structural and mutational analysis of substrate complexation by anthranilate phosphoribosyltransferase from Sulfolobus solfataricus

The metabolic synthesis and degradation of essential nucleotide compounds are primarily carried out by phosphoribosyltransferases (PRT) and nucleoside phosphorylases (NP), respectively. Despite the resemblance of their reactions, five classes of PRTs and NPs exist, where anthranilate PRT (AnPRT) constitutes the only evolutionary link between synthesis and degradation processes. We have characterized the active site of dimeric AnPRT from Sulfolobus solfataricus by elucidating crystal structures of the wild-type enzyme complexed to its two natural substrates anthranilate and 5-phosphoribosyl-1-pyrophosphate/Mg(2+). These bind into two different domains within each protomer and are brought together during catalysis by rotational domain motions as shown by small angle x-ray scattering data. Steady-state kinetics of mutated AnPRT variants address the role of active site residues in binding and catalysis. Results allow the comparative analysis of PRT and pyrimidine NP families and expose related structural motifs involved in nucleotide/nucleoside recognition by these enzyme families.     

The role of strain energy in cycloamylose substrate complexation

The cycloamyloses, a group of cyclic oligosaccharides composed of α-1,4-linked glucose units, provide an opportunity to study the driving forces responsible for enzyme-substrate binding. The cycloamylose substrate binding energy has been attributed to two sources: expulsion of high energy cavity water and release of conformational strain energy. Our studies have shown that release of strain energy plays only a minor role in overall energetics of binding.     

Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]phosphatidylcholine (C₆-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A₂ (PA₂) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C₆-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca⁺⁺ was required for PA₂ catalysis. Identification of the products of hydrolysis showed cleavage at the $\text{sn}$-1 and $\text{sn}$-2 positions for LpL and PA₂, respectively. For PA₂, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.     

Hydrogels of a conducting conjugated polymer as 3-D enzyme electrode

We have utilized the highly conducting poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) aqueous dispersion (PEDOT/PSS) to build a conducting hydrogel matrix. Together with appropriate biomolecules this constitutes a hydrogel bio-electrode. The open hydrogel structure makes diffusion of analytes surrounding the cells into the matrix electrode easier. If enzymes are utilized, osmium is used as mediator between the prosthetic group of the enzyme and the conducting polymer matrix. Osmium also functions as a crosslink point to poly-4-vinylpyridine, which together with the magnesium crosslinked PEDOT/PSS gives a rigid hydrogel. The enzyme Horseradish peroxidase (HRP) was used as a model enzyme to evaluate the enzyme-enhanced electrode. We evaluated the electrode at pH 7, which is the pH choice for many biological systems. From cyclic voltammetry (CV) measurements we deduced that a very low reduction potential was needed to reduce the prosthetic group. Constant potential amperometry were performed to demonstrate the biosensor capabilities. A differential sensitivity of 0.13 A M(-1) cm(-2) through the 0-30 microM concentration range was achieved. Both the biostability and the influence on conductivity, important aspects when for example making nerve- or cell-electrodes, were investigated.